Ligand-functionalized substrates with enhanced binding capacity

ABSTRACT

(b) borne on the porous substrate, a polymer comprising interpolymerized units of at least one monomer consisting of (1) at least one monovalent ethylenically unsaturated group, (2) at least one monovalent ligand functional group selected from acidic groups, basic groups other than guanidino, and salts thereof, and (3) a multivalent spacer group that is directly bonded to the monovalent groups so as to link at least one ethylenically unsaturated group and at least one ligand functional group by a chain of at least six catenated atoms.

STATEMENT OF PRIORITY

This application claims the priority of U.S. Provisional Application No.61/886177 filed Oct. 3, 2013, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

This invention relates to articles comprising ligand-functionalizedsubstrates and, in other aspects, to processes for preparing and usingthe articles.

BACKGROUND

Detection, quantification, isolation, and purification of targetbiomaterials, such as viruses and biomacromolecules (includingconstituents or products of living cells, for example, proteins,carbohydrates, lipids, and nucleic acids) have long been objectives ofinvestigators. Detection and quantification are importantdiagnostically, for example, as indicators of various physiologicalconditions such as diseases. Isolation and purification ofbiomacromolecules are important for therapeutic uses and in biomedicalresearch.

Polymeric materials have been widely used for the separation andpurification of various target biomaterials. Such separation andpurification methods can be based on any of a number of binding factorsor mechanisms including the presence of an ionic group, the size of thetarget biomaterial, a hydrophobic interaction, an affinity interaction,the formation of a covalent bond, and so forth.

Membrane-based technologies, especially in disposable format, arebecoming increasingly important in biopharmaceutical and vaccinemanufacturing processes. Membranes have been used in passive, size-basedseparations (for example, in virus removal applications) and, morerecently, in active filtration (for example, for the removal of minorcontaminants in later stages of purification processes).

Functionalized membranes (including functional polymer-bearingmembranes) have typically suffered from relatively low biomaterialbinding capacities, however, and this has generally limited their use inlarge-scale purifications. Porous beaded chromatography resins (bearingion exchange or other interactive ligand functional groups), rather thanfunctionalized membranes, therefore have been standardly used in“capture-and-elute” type protein purification processes.

SUMMARY

Thus, we recognize that there is a need for ligand-functionalizedsubstrates (particularly, ligand-functionalized membranes) havingrelatively high biomaterial binding capacities. There is an accompanyingneed for ligand-functionalization processes that are relatively simple,cost-effective, and/or efficient (for example, involving relativelyeasily accessible starting materials and/or relatively few processsteps).

Briefly, in one aspect, this invention provides an article that can beused for biomaterial capture. The article comprises

-   -   (a) a porous substrate; and    -   (b) borne on the porous substrate, a polymer comprising        interpolymerized units of at least one monomer consisting of (1)        at least one monovalent ethylenically unsaturated group, (2) at        least one monovalent ligand functional group selected from        acidic groups, basic groups other than guanidino, and salts        thereof, and (3) a multivalent spacer group that is directly        bonded to the monovalent groups so as to link at least one        ethylenically unsaturated group and at least one ligand        functional group by a chain of at least six catenated atoms.        Preferably, the porous substrate is a porous membrane (more        preferably, a porous polymeric membrane). The ligand functional        group(s) are preferably selected from carboxy, phosphono,        phosphato, sulfono, sulfato, boronato, tertiary amino,        quaternary amino, and combinations thereof. The multivalent        spacer group preferably comprises at least one hydrogen bonding        moiety.

It has been discovered that relatively high binding capacity,ligand-functionalized substrates can be prepared by using certainmonomers having a multiatom spacer group between the monomer'spolymerizable group and ligand functional group. The monomers includeligand-functional, ethylenically unsaturated compounds in which at leastone ligand functional group is separated or spaced from at least oneethylenically unsaturated group by a linking chain of at least sixcatenated atoms. When free radically polymerized, such monomers providepolymer bearing ligand functional groups that are separated or spacedfrom the resulting polymer chain or polymer backbone by the linkingchain of at least six catenated atoms.

Surprisingly, the ligand functional groups of such polymers can beespecially effective in interacting with target biomaterials. Poroussubstrates bearing the polymer (preferably, bearing grafted polymer) canexhibit unexpectedly higher biomaterial binding capacities thancorresponding porous substrates bearing polymer derived from monomers inwhich the ligand functional group and the ethylenically unsaturatedgroup are separated by a shorter linking chain.

Binding capacities can be surprisingly further enhanced by including atleast one hydrogen bonding moiety in the linking chain of the monomer.

Such ligand-functionalized substrates can be relatively easily preparedby utilizing free radical polymerization techniques and monomersderivable from readily available starting materials. Theligand-functionalized substrates can be used for various differentapplications, including the capture, binding, or purification ofrelatively neutral or charged biomaterials such as viruses and othermicroorganisms, proteins, cells, endotoxins, acidic carbohydrates,nucleic acids, and the like.

Thus, in at least some embodiments, articles of the invention comprisingthe above-described ligand-functionalized substrates can meet theabove-cited need for ligand-functionalized substrates (particularly,ligand-functionalized membranes) having relatively high biomaterialbinding capacities. In addition, article preparation processes of theinvention, in at least some embodiments, can meet the accompanying needfor ligand-functionalization processes that are relatively simple,cost-effective, and/or efficient (for example, involving relativelyeasily accessible starting materials and/or relatively few processsteps).

In another aspect, this invention further provides a process forpreparing the article of the invention. The process comprises

(a) providing a porous substrate; and

-   -   (b) providing the porous substrate with a polymer comprising        interpolymerized units of at least one monomer consisting of (1)        at least one monovalent ethylenically unsaturated group, (2) at        least one monovalent ligand functional group selected from        acidic groups, basic groups other than guanidino, and salts        thereof, and (3) a multivalent spacer group that is directly        bonded to the monovalent groups so as to link at least one        ethylenically unsaturated group and at least one ligand        functional group by a chain of at least six catenated atoms.

In yet another aspect, this invention also provides a process forcapture or removal of a target biomaterial. The process comprises

-   -   (a) providing at least one article of the invention comprising        at least one filter element; and    -   (b) allowing a moving biological solution containing a target        biomaterial to impinge upon the upstream surface of the filter        element for a time sufficient to effect binding of the target        biomaterial.

In a further aspect, this invention additionally provides a freeradically polymerizable compound or monomer consisting of (a) at leastone monovalent ethylenically unsaturated group, (b) at least onemonovalent ligand functional group selected from phosphorus-containingacidic groups, boron-containing acidic groups, and salts thereof, and(c) a multivalent spacer group that is directly bonded to the monovalentgroups so as to link at least one ethylenically unsaturated group and atleast one ligand functional group by a chain of at least six catenatedatoms. Preferred compounds or monomers include those that can berepresented by a Formula V, which can be obtained by replacing L inFormula I below with L′, a heteroatom-containing group comprising atleast one monovalent ligand functional group selected fromphosphorus-containing acidic groups (preferably, phosphono orphosphato), boron-containing acidic groups (preferably, boronato), andsalts thereof.

DETAILED DESCRIPTION

In the following detailed description, various sets of numerical ranges(for example, of the number of carbon atoms in a particular moiety, ofthe amount of a particular component, or the like) are described, and,within each set, any lower limit of a range can be paired with any upperlimit of a range. Such numerical ranges also are meant to include allnumbers subsumed within the range (for example, 1 to 5 includes 1, 1.5,2, 2.75, 3, 3.80, 4, 5, and so forth).

As used herein, the term and/or means one or all of the listed elementsor a combination of any two or more of the listed elements.

The words preferred and preferably refer to embodiments of the inventionthat may afford certain benefits under certain circumstances. Otherembodiments may also be preferred, however, under the same or othercircumstances. Furthermore, the recitation of one or more preferredembodiments does not imply that other embodiments are not useful, and isnot intended to exclude other embodiments from the scope of theinvention.

The term comprises and variations thereof do not have a limiting meaningwhere these terms appear in the description and claims.

As used herein, a, an, the, at least one, and one or more are usedinterchangeably.

The above Summary of the Invention section is not intended to describeevery embodiment or every implementation of the invention. The detaileddescription that follows more particularly describes illustrativeembodiments. Throughout the detailed description, guidance is providedthrough lists of examples, which examples can be used in variouscombinations. In each instance, a recited list serves only as arepresentative group and should not be interpreted as being an exclusivelist.

Definitions

As used in this patent application:

“boronato” means a monovalent group of formula —B(OH)2;

“carbonylimino” means a divalent group or moiety of formula —(CO)NR—,where R is hydrogen, alkyl (for example, selected from alkyl groupshaving from one to about four carbon atoms), or aryl (preferably,hydrogen);

“carboxy” means a monovalent group of formula —COOH;

“catenated atom” means an in-chain atom (rather than an atom of a chainsubstituent);

“catenated heteroatom” means an atom other than carbon (for example,oxygen, nitrogen, or sulfur) that replaces one or more carbon atoms in acarbon chain (for example, so as to form a carbon-heteroatom-carbonchain or a carbon-heteroatom-heteroatom-carbon chain);

“ethylenically unsaturated” means a group of formula —CY═CH2 where Y ishydrogen, alkyl, cycloalkyl, or aryl;

“guanidino” means a monovalent group of formula R″₂N—C(═NR″)NH— whereeach R″ is independently hydrogen, hydrocarbyl, heterohydrocarbyl, or acombination thereof, and where any two or more R″ groups optionally canbe bonded together to form a ring structure (preferably, each R″ isindependently hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl,heteroaryl, or a combination thereof; more preferably, each R″ isindependently hydrogen, alkyl, cycloalkyl, aryl, or a combinationthereof);

“heteroatom” means an atom other than carbon or hydrogen;

“hydrogen bond acceptor” means a heteroatom selected from oxygen,nitrogen, and sulfur that has a lone electron pair;

“hydrogen bond donor” means a moiety consisting of a hydrogen atomcovalently bonded to a heteroatom selected from oxygen, nitrogen, andsulfur;

“hydrogen bonding moiety” means a moiety comprising at least onehydrogen bond donor and at least one hydrogen bond acceptor;

“hydroxy” means a monovalent group of formula —OH;

“iminocarbonylimino” means a divalent group or moiety of formula—N(R)—C(O)—N(R)—, wherein each R is independently hydrogen, alkyl (forexample, selected from alkyl groups having from one to about four carbonatoms), or aryl (preferably, at least one R is hydrogen; morepreferably, both are hydrogen);

“iminothiocarbonylimino” means a divalent group or moiety of formula—N(R)—C(S)—N(R)—, wherein each R is independently hydrogen, alkyl (forexample, selected from alkyl groups having from one to about four carbonatoms), or aryl (preferably, at least one R is hydrogen; morepreferably, both are hydrogen);

“isocyanato” means a monovalent group of formula —N═C═O;

“oxycarbonylimino” means a divalent group or moiety of formula—O—C(O)—N(R)—, wherein R is hydrogen, alkyl (for example, selected fromalkyl groups having from one to about four carbon atoms), or aryl(preferably, hydrogen);

“oxythiocarbonylimino” means a divalent group or moiety of formula—O—C(S)—N(R)—, wherein R is hydrogen, alkyl (for example, selected fromalkyl groups having from one to about four carbon atoms), or aryl(preferably, hydrogen);

“phosphato” means a monovalent group of formula —OPO₃H₂;

“phosphono” means a monovalent group of formula —PO₃H₂;

“quaternary amino” means a monovalent group of formula —NR′₃ ⁺, whereeach R′ is independently hydrocarbyl, heterohydrocarbyl, or acombination thereof, and where any two or more R′ groups optionally canbe bonded together to form a ring structure (preferably, each R′ isindependently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl,heteroaryl, or a combination thereof; more preferably, each R′ isindependently alkyl, cycloalkyl, aryl, or a combination thereof);

“sulfato” means a monovalent group of formula —OSO₃H;

“sulfono” means a monovalent group of formula —SO₃H;

“tertiary amino” means a monovalent group of formula —NR′₂, where eachR′ is independently hydrocarbyl, heterohydrocarbyl, or a combinationthereof, and where any two or more R′ groups optionally can be bondedtogether to form a ring structure (preferably, each R′ is independentlyalkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, heteroaryl, or acombination thereof; more preferably, each R′ is independently alkyl,cycloalkyl, aryl, or a combination thereof); and

“thiocarbonylimino” means a divalent group or moiety of formula—(CS)NR—, where R is hydrogen, alkyl (for example, selected from alkylgroups having from one to about four carbon atoms), or aryl (preferably,hydrogen).

Monomers

Monomers suitable for use in preparing the article of the inventioninclude those that consist of (a) at least one monovalent ethylenicallyunsaturated group, (b) at least one monovalent ligand functional groupselected from acidic groups, basic groups other than guanidino, andsalts thereof, and (c) a multivalent spacer group that is directlybonded to the monovalent groups so as to link at least one ethylenicallyunsaturated group and at least one ligand functional group by a chain ofat least six catenated atoms. Preferably, the monomer(s) contain onlymoieties other than guanidino moieties. The monomers can be in a neutralstate but can also be negatively (if acidic) or positively (if basic)charged under some pH conditions. The monomers can be permanentlycharged when the ligand functional group is in the form of a salt (forexample, when the ligand functional group comprises quaternary ammoniumor N-alkylpyridinium).

The monovalent ethylenically unsaturated group (as defined above) of themonomer(s) can be represented by the formula —CY═CH₂, wherein Y ishydrogen, alkyl, cycloalkyl, or aryl.

Preferred ethylenically unsaturated groups include ethenyl,1-alkylethenyl, and combinations thereof (that is, Y is preferablyhydrogen or alkyl; more preferably, Y is hydrogen or C₁ to C₄ alkyl;most preferably, Y is hydrogen or methyl). The monomer(s) can comprise asingle ethylenically unsaturated group or multiple ethylenicallyunsaturated groups (for example, two or three or up to as many as 6),which can be the same or different in nature (preferably, the same). Themonomer(s) preferably have only one ethylenically unsaturated group.

The monovalent ligand functional group of the monomer(s) can be selectedfrom acidic groups, basic groups other than guanidino, and saltsthereof. Suitable ligand functional groups include those that exhibit atleast a degree of acidity or basicity (which can range from relativelyweak to relatively strong), as well as salts thereof. Such ligandfunctional groups include those commonly utilized as ion exchange ormetal chelate type ligands.

Useful ligand functional groups include heterohydrocarbyl groups andother heteroatom-containing groups. For example, useful acidic or basicligand functional groups can comprise one or more heteroatoms selectedfrom oxygen, nitrogen, sulfur, phosphorus, boron, and the like, andcombinations thereof. Useful salts of acidic groups include those havingcounter ions selected from alkali metal (for example, sodium orpotassium), alkaline earth metal (for example, magnesium or calcium),ammonium, and tetraalkylammonium ions, and the like, and combinationsthereof. Useful salts of basic groups include those having counter ionsselected from halide (for example, chloride or bromide), carboxylate,nitrate, phosphate, sulfate, bisulfate, methyl sulfate, and hydroxideions, and the like, and combinations thereof.

The monomer(s) can comprise a single ligand functional group or multipleligand functional groups (for example, two or three or up to as many as6), which can be the same or different in nature (preferably, the same).The ligand functional group(s) are preferably selected from carboxy,phosphono, phosphato, sulfono, sulfato, boronato, tertiary amino,quaternary amino, and combinations thereof. More preferred ligandfunctional group(s) include carboxy, phosphono, sulfono, tertiary amino,quaternary amino, and combinations thereof.

The multivalent spacer group of the monomer(s) can be directly bonded tothe monovalent groups so as to link at least one ethylenicallyunsaturated group and at least one ligand functional group by a chain ofat least six catenated atoms. Thus, the chain can comprise 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, or more catenated atoms (for example,including up to as many as 40 or 50). The chain preferably comprises atleast seven catenated atoms (more preferably, at least eight; mostpreferably, at least nine, ten, eleven, or twelve) and/or comprises nomore than about 30 catenated atoms (more preferably, no more than about25; even more preferably, no more than about 20; most preferably, nomore than about 16).

Although not wishing to be bound by theory, the length of the chain maycontribute to adoption of helical or partially helical conformations bythe polymer backbone (formed through monomer polymerization). When thechain is relatively short (for example, less than about six catenatedatoms), ionic repulsion between charged ligand functional groups mayforce the polymer backbone into a random coil type conformation. Aschain length increases, adoption of helical conformations may becomepossible and may be maximized at chain lengths of about 8-14 catenatedatoms. A helical conformation of substrate-grafted polymer mayfacilitate presentation of the ligand functional group(s) forinteraction with a target biomaterial.

Preferred multivalent spacer groups comprise at least one hydrogenbonding moiety, which is defined above as a moiety comprising at leastone hydrogen bond donor and at least one hydrogen bond acceptor (both ofwhich are heteroatom-containing, as described above). Thus, preferredmultivalent spacer groups include heteroatom-containing hydrocarbongroups (more preferably, catenated heteroatom-containing hydrocarbongroups). More preferred spacer groups comprise at least two hydrogenbonding moieties or comprise at least one hydrogen bonding moiety and atleast one hydrogen bond acceptor that is distinct from (not part of) thehydrogen bonding moiety.

Preferred hydrogen bonding moieties include those that comprise at leasttwo hydrogen bond donors (for example, donors such as imino, thio, orhydroxy), at least two hydrogen bond acceptors (for example, acceptorsin the form of carbonyl, carbonyloxy, or ether oxygen), or both. Forexample, an iminocarbonylimino moiety (having two N-H donors and atleast two acceptors in the form of two lone electron pairs on carbonyl)can sometimes be preferred over a single iminocarbonyl moiety. Preferredspacer groups include those that comprise at least oneiminocarbonylimino moiety (more preferably, in combination with at leastone acceptor such as carbonyloxy), at least two iminocarbonyl moieties,or a combination thereof.

The hydrogen bond donor and hydrogen bond acceptor of the hydrogenbonding moiety can be adjacent (directly bonded) to each other or can benon-adjacent (preferably, adjacent or separated by a chain of no morethan about 4 catenated atoms; more preferably, adjacent). Theheteroatoms of the hydrogen bond donor and/or hydrogen bond acceptor canbe located in the chain of catenated atoms of the spacer group or,alternatively, can be located in chain substituents.

Although hydrogen bond donors can also function as hydrogen bondacceptors (through a lone electron pair of the donor's heteroatom), thehydrogen bonding moiety preferably comprises distinct donor and acceptormoieties. This can facilitate intramolecular (intermonomer) hydrogenbond formation. Although not wishing to be bound by theory, suchintramolecular hydrogen bonds between adjacent monomer repeat units inthe polymer molecule may contribute to at least a degree of multivalentspacer group stiffening, which may facilitate presentation of the ligandfunctional group(s) for interaction with a target biomaterial.

Preferred hydrogen bonding moieties include carbonylimino,thiocarbonylimino, iminocarbonylimino, iminothiocarbonylimino,oxycarbonylimino, oxythiocarbonylimino, and the like, and combinationsthereof. More preferred hydrogen bonding moieties include carbonylimino,iminocarbonylimino, oxycarbonylimino, and combinations thereof (mostpreferably, carbonylimino, iminocarbonylimino, and combinationsthereof). Preferred multivalent spacer groups include those that aredivalent, trivalent, or tetravalent (more preferably, divalent ortrivalent; most preferably, divalent).

A class of useful monomers includes those represented by the followinggeneral formula

CH₂═CR¹—C(═O)—X—R²—[Z—R²]_(n)—L   (I)

wherein

-   -   R¹ is selected from hydrogen, alkyl, cycloalkyl, aryl, and        combinations thereof;    -   each R² is independently selected from hydrocarbylene,        heterohydrocarbylene, and combinations thereof;    -   X is —O— or —NR³—, where R³ is selected from hydrogen,        hydrocarbyl, heterohydrocarbyl, and combinations thereof;    -   Z is heterohydrocarbylene comprising at least one hydrogen bond        donor, at least one hydrogen bond acceptor, or a combination        thereof;    -   n is an integer of 0 or 1; and

L is a heteroatom-containing group comprising at least one monovalentligand functional group selected from acidic groups, basic groups otherthan guanidino, and salts thereof.

Preferably, R¹ is hydrogen or alkyl (more preferably, hydrogen or C₁ toC₄ alkyl; most preferably, hydrogen or methyl); each R² is independentlyhydrocarbylene (more preferably, independently alkylene); X is —O— orwhere R³ is hydrogen; Z is heterohydrocarbylene comprising at least onemoiety selected from carbonyl, carbonylimino, carbonyloxy, ether oxygen,thiocarbonylimino, iminocarbonylimino, iminothiocarbonylimino,oxycarbonylimino, oxythiocarbonylimino, and combinations thereof (morepreferably, selected from carbonyl, carbonylimino, carbonyloxy, etheroxygen, iminocarbonylimino, oxycarbonylimino, and combinations thereof;even more preferably, selected from carbonylimino, carbonyloxy, etheroxygen, iminocarbonylimino, and combinations thereof; most preferably,selected from carbonylimino, iminocarbonylimino, and combinationsthereof); n is an integer of 1; and/or L is a heteroatom-containinggroup comprising at least one ligand functional group selected fromcarboxy, phosphono, phosphato, sulfono, sulfato, boronato, tertiaryamino, quaternary amino, and combinations thereof (more preferably,selected from carboxy, phosphono, sulfono, tertiary amino, quaternaryamino, and combinations thereof).

Such monomers can be prepared by known synthetic methods or by analogyto known synthetic methods. For example, amino group-containingcarboxylic, sulfonic, or phosphonic acids can be reacted withethylenically unsaturated compounds that comprise at least one groupthat is reactive with an amino group. Similarly, ligand functionalgroup-containing compounds that also contain a hydroxy group can bereacted with ethylenically unsaturated compounds that comprise at leastone group that is reactive with a hydroxy group, optionally in thepresence of a catalyst. Preferred monomers are(meth)acryloyl-functional. (As used herein, the term“(meth)acryloyl-functional” refers to acryloyl-functional and/ormethacryloyl-functional; similarly, the term “(meth)acrylate” refers toan acrylate and/or a methacrylate).

Representative examples of useful monomers include those derived fromthe reaction of an alkenyl azlactone of general Formula II

or an ethylenically unsaturated isocyanate of general Formula III

CH₂═C(R′)—C(═O)—X—R²—N═C═O   (III)

with a ligand functional group-containing compound of general Formula IV

H—X—R²—L   (IV)

to produce monomer of general Formula I (wherein R¹, X, R², and L inFormulas II, III, and/or IV are as defined above for Formula I).Representative examples of useful alkenyl azlactones of Formula IIinclude 4,4-dimethyl-2-vinyl-4H-oxazol-5-one (vinyldimethylazlactone,VDM), 2-isopropenyl-4H-oxazol-5-one,4,4-dimethyl-2-isopropenyl-4H-oxazol-5-one,2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,4,4-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,4,5-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one, and the like, andcombinations thereof. Representative examples of ethylenicallyunsaturated isocyanates of general Formula III include 2-isocyanatoethyl(meth)acrylate (IEM or IEA), 3-isocyanatopropyl (meth)acrylate,4-isocyanatocyclohexyl (meth)acrylate, and the like, and combinationsthereof.

Representative examples of useful ligand functional group-containingcompounds of general Formula IV include amino group-containingcarboxylic, sulfonic, boronic, and phosphonic acids and combinationsthereof. Useful amino carboxylic acids include α-amino acids (L-, D-, orDL-α-amino acids) such as glycine, alanine, valine, proline, serine,phenylalanine, histidine, tryptophan, asparagine, glutamine,N-benzylglycine, N-phenylglycine, sarcosine, and the like; β-aminoacidssuch as β-alanine, β-homoleucine, β-homoglutamine, β-homophenylalanine,and the like; other α,ω-aminoacids such as γ-aminobutyric acid,6-aminohexanoic acid, 11-aminoundecanoic acid, peptides (such asdiglycine, triglycine, tetraglycine, as well as other peptidescontaining a mixture of different aminoacids), and the like; andcombinations thereof. Useful amino sulfonic acids includeaminomethanesulfonic acid, 2-aminoethanesulfonic acid (taurine),3-amino-1-propanesulfonic acid, 6-amino-1-hexanesulfonic acid, and thelike, and combinations thereof. Useful aminoboronic acids includem-aminophenylboronic acid, p-aminophenylboronic acid, and the like, andcombinations thereof. Useful aminophosphonic acids include1-aminoethylphosphonic acid, 2-aminoethylphosphonic acid,3-aminopropylphosphonic acid, and the like, and combinations thereof.Useful compounds of Formula IV containing more than one ligandfunctional group include aspartic acid, glutamic acid, α-aminoadipicacid, iminodiacetic acid, N_(α),N_(α)-bis(carboxymethyl)lysine, cysteicacid, N-phosphonomethylglycine, and the like, and combinations thereof.

Representative examples of other useful ligand functionalgroup-containing compounds of general Formula IV include compoundscomprising a hydroxy group and an acidic group. Specific examplesinclude glycolic acid, lactic acid, 6-hydroxyhexanoic acid, citric acid,2hydroxyethylsulfonic acid, 2-hydroxyethylphosphonic acid, and the like,and combinations thereof.

Representative examples of still other useful ligand functionalgroup-containing compounds of general Formula IV include compoundscomprising at least one amino or hydroxy group and at least one basicgroup such as a tertiary or quaternary amino group. Specific examplesinclude 2-(dimethylamino)ethylamine, 3-(diethylamino)propylamine,6-(dimethylamino)hexylamine, 2-aminoethyltrimethylammonium chloride,3-aminopropyltrimethylammonium chloride, 2-(dimethylamino)ethanol,3-(dimethylamino)-1-propanol, 6-(dimethylamino)-1-hexanol,1-(2-aminoethyl)pyrrolidine, 2-[2-(dimethylamino)ethoxy]ethanol,histamine, 2-aminomethylpyridine, 4-aminomethylpyridine,4-aminoethylpyridine, and the like, and combinations thereof.

Many of the above-described ligand functional group-containing compoundsof general Formula IV are commercially available. Still other usefulligand functional group-containing compounds of general Formula IV canbe prepared by common synthetic procedures. For example, variousdiamines or aminoalcohols can be reacted with one equivalent of a cyclicanhydride to produce an intermediate ligand functional group-containingcompound comprising a carboxyl group and an amino or hydroxy group.

Useful monomers can also be prepared by the reaction of ligandfunctional group-containing compounds of general Formula IV withethylenically unsaturated acyl halides (for example, (meth)acryloylchloride). In addition, useful monomers can be prepared by reaction ofhydroxy- or amine-containing (meth)acrylate or (meth)acrylamide monomerswith a cyclic anhydride to produce carboxyl group-containing monomers.

Preferred monomers include monomers prepared from the reaction ofalkenyl azlactones with aminocarboxylic acids, monomers prepared fromthe reaction of alkenyl azlactones with aminosulfonic acids, monomersprepared from the reaction of alkenyl azlactones with ligand functionalgroup-containing compounds comprising a primary amino group and atertiary or quaternary amino group, monomers prepared from the reactionof ethylenically unsaturated isocyanates with aminocarboxylic acids,monomers prepared from the reaction of ethylenically unsaturatedisocyanates with aminosulfonic acids, monomers prepared from thereaction of ethylenically unsaturated isocyanates with ligand functionalgroup-containing compounds comprising a primary or secondary amino groupand a tertiary or quaternary amino group, and combinations thereof.

In preparing the article of the invention, the above-described monomersgenerally (and preferably) can be homopolymerized. A skilled artisanwill recognize, however, that the monomers can be copolymerized withother monomers (hereinafter, termed “comonomers”; for example,comonomers having shorter spacer groups or comonomers comprising othertypes of ligands or even comonomers that are ligand-free) in order toadjust binding capacity and/or to achieve special properties, providedthat the type and degree of binding capacity desired for a particularapplication can be achieved.

For example, the monomer(s) optionally can be copolymerized with one ormore hydrophilic comonomer(s) comprising at least one alkenyl group(preferably, a (meth)acryloyl group) and a hydrophilic group (includingpoly(oxyalkylene) groups) in order to impart a degree of hydrophilicityto the porous substrate. Suitable hydrophilic comonomers includeacrylamide, dimethylacrylamide, hydroxyethyl(meth)acrylate,hydroxypropyl(meth)acrylate, polyethyleneglycolmono(meth)acrylate,2-hydroxyethylacrylamide, N-vinylpyrrolidone, and the like, andcombinations thereof.

Optionally, the monomer(s) can be copolymerized with one or more(meth)acryloyl comonomer(s) containing at least two free radicallypolymerizable groups. Such multifunctional (meth)acryloyl comonomer(s)(including multifunctional (meth)acrylate(s) and (meth)acrylamide(s))can be incorporated in a blend of polymerizable monomer(s) generally inonly relatively small amounts (for example, from about 0.1 to about 5percent by weight, based upon the total weight of monomer(s) andcomonomer(s)) to impart a degree of branching and/or relatively lightcrosslinking to a resulting copolymer. Higher amounts can be used forcertain applications, but it should be understood that the use of higheramounts may reduce binding capacity for a target biomaterial.

Useful multifunctional (meth)acryloyl comonomers includedi(meth)acrylates, tri(meth)acrylates, tetra(meth)acrylates,multifunctional (meth)acrylamides, and the like, and combinationsthereof. Such multifunctional (meth)acryloyl comonomers includeethyleneglycol di(meth)acrylate, 1,6-hexanediol di(meth)acrylate,poly(ethylene glycol) di(meth)acrylates, polybutadiene di(meth)acrylate,polyurethane di(meth)acrylates, propoxylated glycerin tri(meth)acrylate,methylenebisacrylamide, ethylenebisacrylamide,hexamethylenebisacrylamide, diacryloylpiperazine, and the like, andcombinations thereof

Article Preparation and Use

Polymerization of the monomer(s) can be carried out by using knowntechniques. For example, the polymerization can be initiated with eithera thermal initiator or a photoinitiator (preferably, a photoinitiator).Essentially any conventional free radical initiator can be used togenerate the initial radical. Examples of suitable thermal initiatorsinclude peroxides such as benzoyl peroxide, dibenzoyl peroxide, dilaurylperoxide, cyclohexane peroxide, methyl ethyl ketone peroxide,hydroperoxides (for example, tert-butyl hydroperoxide and cumenehydroperoxide), dicyclohexyl peroxydicarbonate, t-butyl perbenzoate;2,2,-azo-bis(isobutyronitrile); and the like; and combinations thereof.Examples of commercially available thermal initiators include initiatorsavailable from DuPont Specialty Chemical (Wilmington, Del.) under theVAZO trade designation including VAZO™ 67(2,2′-azo-bis(2-methylbutyronitrile)), VAZO™ 64(2,2′-azo-bis(isobutyronitrile)), and VAZO™ 52(2,2′-azo-bis(2,2-dimethylvaleronitrile)), as well as Lucidol™ 70(benzoylperoxide) available from Elf Atochem North America,Philadelphia, Pa.

Useful photoinitiators include benzoin ethers such as benzoin methylether and benzoin isopropyl ether; substituted acetophenones such as 2,2-dimethoxyacetophenone available as Irgacure™ 651 photoinitiator (CibaSpecialty Chemicals), 2,2 dimethoxy-2-phenyl-1-phenylethanone availableas Esacure™ KB-1 photoinitiator (Sartomer Co.; West Chester, Pa.),1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-oneavailable as Irgacure™ 2959 (Ciba Specialty Chemicals), anddimethoxyhydroxyacetophenone; substituted α-ketols such as2-methyl-2-hydroxy propiophenone; aromatic sulfonyl chlorides such as2-naphthalene-sulfonyl chloride; photoactive oximes such as1-phenyl-1,2-propanedione-2-(O-ethoxy-carbonyl)oxime; and the like; andcombinations thereof. Particularly preferred among these are thesubstituted acetophenones (especially1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one,Irgacure™ 2959, due to its water solubility). A particularly usefulpolymerizable photoinitiator is a 1:1 adduct of2-vinyl-4,4-dimethylazlactone and Irgacure™ 2959, which can be preparedessentially as described in Example 1 of U.S. Pat. No. 5,506,279 (Babuet al.), the description of which preparation is incorporated herein byreference.

Other useful photoinitiators include hydrogen-abstracting (Type II)photoinitiators such as benzophenone, 4-(3-sulfopropyloxy)benzophenonesodium salt, Michler's ketone, benzil, anthraquinone,5,12-naphthacenequinone, aceanthracenequinone,benz(A)anthracene-7,12-dione, 1,4-chrysenequinone,6,13-pentacenequinone, 5,7,12,14-pentacenetetrone, 9-fluorenone,anthrone, xanthone, thioxanthone, 2-(3-sulfopropyloxy)thioxanthen-9-one,acridone, dibenzosuberone, acetophenone, chromone, and the like, andcombinations thereof.

The initiator can be used in an amount effective to initiate freeradical polymerization of the monomer(s). Such amount will varydepending upon, for example, the type of initiator and polymerizationconditions utilized. The initiator generally can be used in amountsranging from about 0.01 part by weight to about 5 parts by weight, basedupon 100 parts total monomer.

The polymerization solvent can be essentially any solvent that cansubstantially dissolve (or, in the case of emulsion or suspensionpolymerizations, disperse or suspend) the monomer(s) (and comonomer(s),if used). In many embodiments, the solvent can be water or awater/water-miscible organic solvent mixture. The ratio of water toorganic solvent can vary widely, depending upon monomer solubility. Withsome monomers, the ratio typically can be greater than 1:1(volume/volume) water to organic solvent (preferably, greater than 5:1;more preferably, greater than 7:1). With other monomers, a higherproportion of organic solvent (even up to 100 percent) can be preferred(with some alcohols especially).

Any such water-miscible organic solvent preferably has no groups thatwould retard polymerization. In some embodiments, the water-misciblesolvents can be protic group-containing organic liquids such as thelower alcohols having 1 to 4 carbon atoms, lower glycols having 2 to 6carbon atoms, and lower glycol ethers having 3 to 6 carbon atoms and 1to 2 ether linkages. In some embodiments, higher glycols such aspoly(ethylene glycol) can be used. Specific examples include methanol,ethanol, isopropanol, n-butanol, t-butyl alcohol, ethylene glycol,methoxyethanol, ethoxyethanol, propoxyethanol, butoxyethanol, methylcarbitol, ethyl carbitol, and the like, and combinations thereof.

In other embodiments, non-protic water-miscible organic solvents can beused. Such solvents include aliphatic esters (for example, methoxyethylacetate, ethoxyethyl acetate, propoxyethyl acetate, butoxyethyl acetate,and triethyl phosphate), ketones (for example, acetone, methyl ethylketone, and methyl propyl ketone), and sulfoxides (for example, dimethylsulfoxide).

The monomer concentration in the polymerization solvent can vary,depending upon a number of factors including, but not limited to, thenature of the monomer or monomers, the extent of polymerization desired,the reactivity of the monomer(s), and the solvent used. Typically, themonomer concentration can range from about 0.1 weight percent (wt %) toabout 60 wt% (preferably, from about 1 wt % to about 40 wt %; morepreferably, from about 5 wt % to about 30 wt %), based upon the totalweight of monomer and solvent.

An aqueous monomer mixture optionally can be formulated with relativelyhigher levels of multifunctional (crosslinking) monomers or comonomers(for example, from about 5 percent (%) by weight up to about 90% byweight, based upon the total weight of monomer(s) and comonomer(s)) andpolymerized as a suspension or dispersion in a nonpolar, immiscibleorganic solvent, optionally in the presence of added porogen(s), toproduce crosslinked, porous particles comprising the instant monomer(s).Such methods are well known and are described, for example, in U.S. Pat.No. 7,098,253 (Rasmussen et al.), U.S. Pat. No. 7,674,835 (Rasmussen etal.), U.S. Pat. No. 7,647,836 (Rasmussen et al.), and U.S. Pat. No.7,683,100 (Rasmussen et al.), the descriptions of which methods areincorporated herein by reference.

If desired, the polymerization can be carried out in the presence of aporous substrate, so as to form an article comprising a porous substratebearing the resulting polymer. For example, an imbibing or coatingsolution comprising the monomer(s), any comonomer(s), initiator(s), andsolvent(s) can be imbibed by or coated (or otherwise deposited) on aporous substrate. The porous substrate can be in essentially any formsuch as particles, fibers, films, webs, membranes, sponges, or sheets.Suitable porous substrates can be organic, inorganic, or a combinationthereof (preferably, organic; more preferably, polymeric). Suitableporous substrates include porous particles, porous membranes, porousnonwoven webs, porous woven webs, porous sponges, porous fibers, and thelike, and combinations thereof. Preferred porous substrates includeporous membranes (more preferably, porous polymeric membranes; mostpreferably, porous polyamide membranes) and combinations thereof.

For example, the porous substrate can be formed from any suitablethermoplastic polymeric material. Suitable polymeric materials includepolyolefins, poly(isoprenes), poly(butadienes), fluorinated polymers,chlorinated polymers, polyamides, polyimides, polyethers, poly(ethersulfones), poly(sulfones), poly(vinyl acetates), polyesters such aspoly(lactic acid), copolymers of vinyl acetate such aspoly(ethylene)-co-poly(vinyl alcohol), poly(phosphazenes), poly(vinylesters), poly(vinyl ethers), poly(vinyl alcohols), poly(carbonates), andthe like, and combinations thereof.

In some embodiments, the thermoplastic polymer can be surface treated,such as by plasma discharge or by use of a primer, to provide suitablefunctionality to the surface of the porous substrate. Surface treatmentcan provide functional groups such as hydroxy groups that can improvewetting by the monomer solution. One such useful plasma treatment isdescribed in U.S. Pat. No. 7,125,603 (David et al.).

Suitable polyolefins include poly(ethylene), poly(propylene),poly(l-butene), copolymers of ethylene and propylene, alpha olefincopolymers (such as copolymers of ethylene or propylene with 1-butene,1-hexene, 1-octene, and 1-decene), poly(ethylene-co-1-butene),poly(ethylene-co-1-butene-co-1-hexene), and the like, and combinationsthereof.

Suitable fluorinated polymers include poly(vinyl fluoride),poly(vinylidene fluoride), copolymers of vinylidene fluoride (such aspoly(vinylidene fluoride-co-hexafluoropropylene)), copolymers ofchlorotrifluoroethylene (such aspoly(ethylene-co-chlorotrifluoroethylene)), and the like, andcombinations thereof.

Suitable polyamides include poly(iminoadipolyliminohexamethylene),poly(iminoadipolyliminodecamethylene), polycaprolactam, and the like,and combinations thereof. Suitable polyimides includepoly(pyromellitimide), and the like, and combinations thereof.

Suitable poly(ether sulfones) include poly(diphenylether sulfone),poly(diphenylsulfone-co-diphenylene oxide sulfone), and the like, andcombinations thereof.

Suitable copolymers of vinyl acetate include poly(ethylene-co-vinylacetate), such copolymers in which at least some of the acetate groupshave been hydrolyzed to afford various poly(vinyl alcohols), and thelike, and combinations thereof.

A preferred porous substrate is a microporous membrane such as athermally-induced phase separation (TIPS) membrane. TIPS membranes areoften prepared by forming a solution of a thermoplastic material and asecond material above the melting point of the thermoplastic material.Upon cooling, the thermoplastic material crystallizes and phaseseparates from the second material. The crystallized material is oftenstretched. The second material is optionally removed either before orafter stretching. Microporous membranes are further described in U.S.Pat. No. 4,539,256 (Shipman); U.S. Pat. No. 4,726,989 (Mrozinski); U.S.Pat. No. 4,867,881 (Kinzer); U.S. Pat. No. 5,120,594 (Mrozinski); U.S.Pat. No. 5,260,360 (Mrozinski); and U.S. Pat. No. 5,962,544 (Waller,Jr.). Some exemplary TIPS membranes comprise poly(vinylidene fluoride)(PVDF), polyolefins such as poly(ethylene) or poly(propylene),vinyl-containing polymers or copolymers such as ethylene-vinyl alcoholcopolymers and butadiene-containing polymers or copolymers, andacrylate-containing polymers or copolymers. For some applications, aTIPS membrane comprising PVDF can be particularly desirable. TIPSmembranes comprising PVDF are further described in U.S. Pat. No.7,338,692 (Smith et al.).

In many embodiments, the porous substrate can have an average pore sizethat is typically greater than about 0.2 micrometers in order tominimize size exclusion separations, minimize diffusion constraints, andmaximize surface area and separation based on binding of a targetbiomaterial. Generally, the pore size can be in the range of 0.1 to 10micrometers (preferably, 0.5 to 3 micrometers; most preferably, 0.8 to 2micrometers when used for binding of viruses and/or proteins). Theefficiency of binding other target biomaterials can confer differentoptimal ranges. In an exemplary embodiment, the porous substrate cancomprise a nylon microporous film or sheet (for example, a microporousmembrane), such as those described in U.S. Pat. No. 6,056,529 (Meyeringet al.), U.S. Pat. No. 6,267,916 (Meyering et al.), U.S. Pat. No.6,413,070 (Meyering et al.), U.S. Pat. No. 6,776,940 (Meyering et al.),U.S. Pat. No. 3,876,738 (Marinacchio et al.), U.S. Pat. No. 3,928,517(Knight et al.), U.S. Pat. No. 4,707,265 (Barnes, Jr. et al.), and U.S.Pat. No. 5,458,782 (Hou et al.).

In other embodiments, the porous substrate can be a nonwoven web, whichcan include nonwoven webs manufactured by any of the commonly knownprocesses for producing nonwoven webs. As used herein, the term“nonwoven web” refers to a fabric that has a structure of individualfibers or filaments that are randomly and/or unidirectionally interlaidin a mat-like fashion.

For example, the fibrous nonwoven web can be made by wet laid, carded,air laid, spunlaced, spunbonding, or melt-blowing techniques, orcombinations thereof. Spunbonded fibers are typically small diameterfibers that are formed by extruding molten thermoplastic polymer asfilaments from a plurality of fine, usually circular capillaries of aspinneret, with the diameter of the extruded fibers being rapidlyreduced. Meltblown fibers are typically formed by extruding moltenthermoplastic material through a plurality of fine, usually circular,die capillaries as molten threads or filaments into a high velocity,usually heated gas (for example, air) stream, which attenuates thefilaments of molten thermoplastic material to reduce their diameter.Thereafter, the meltblown fibers are carried by the high velocity gasstream and are deposited on a collecting surface to form a web ofrandomly-dispersed, meltblown fibers. Any of the nonwoven webs can bemade from a single type of fiber or from two or more fibers that differin the type of thermoplastic polymer and/or thickness.

Further details of manufacturing methods of useful nonwoven webs havebeen described by Wente in “Superfine Thermoplastic Fibers,” 48 Indus.Eng. Chem. 1342 (1956) and by Wente et al. in “Manufacture Of SuperfineOrganic Fibers,” Naval Research Laboratories Report No. 4364 (1954).

Following polymerization, washing, and drying, typical total weightgains by the porous substrate generally can be in the range of about 5percent (%) to about 30% (preferably, in the range of about 10% to about25%; more preferably, in the range of about 12% to about 20%).Polymerization of the monomer(s) in the presence of a porous substratecan produce a polymer-bearing porous substrate. The polymer can be inthe form of a coating or, in preferred embodiments, the polymer can begrafted (covalently bonded) to the surface of the porous substrate. (Ifdesired, the polymerization can be carried out separately and theresulting polymer then coated (optionally in the presence of suitablecrosslinker) or grafted or otherwise applied to the porous substrate,but this is generally less preferred.)

In an exemplary method, the monomer(s) can be free radically polymerizedand grafted to the surface of a porous substrate in the presence of aType II photoinitiator, as described in International Patent ApplicationNo. US2013/042330 (3M Innovative Properties Co.), the description ofwhich method is incorporated herein by reference. Alternatively, themonomer(s) can be free radically polymerized and grafted to a poroussubstrate comprising a crosslinked copolymer layer, the copolymercomprising photoinitiator-containing monomer units, as described in U.S.Provisional Patent Application No. 61/706,288 (Rasmussen et al.), thedescription of which method is incorporated herein by reference. Inaddition, the monomer(s) can be free radically polymerized and graftedto a porous substrate comprising a crosslinked polymer primer layer, asdescribed in U.S. Patent Application Publication No. 2012/0252091 A1(Rasmussen et al.), the description of which method is incorporatedherein by reference.

In yet another exemplary method, the monomer(s) can be free radicallypolymerized and grafted to a porous particle, as described in U.S.Patent Application Publication No. 2011/0100916 A1 (Shannon et al.), thedescription of which method is incorporated herein by reference.

The coated or grafted polymer (which is ligand-functional polymer due tothe presence of the ligand functional groups of the monomer(s)) canalter the original nature of the porous substrate. The resultingpolymer-bearing porous substrates (ligand-functionalized poroussubstrates) can retain many of the advantages of the original poroussubstrate (for example, mechanical and thermal stability, porosity, andso forth) but can also exhibit enhanced affinity for biomaterials suchas viruses, proteins, and the like. Porous substrates bearing theligand-functional polymer can be particularly useful as filter media forthe selective binding and removal of target biomaterials or biologicalspecies (including relatively neutral or charged biomaterials such asviruses and other microorganisms, acidic carbohydrates, proteins,nucleic acids, endotoxins, bacteria, cells, cellular debris, and thelike) from biological samples. Articles comprising the polymer-bearingporous substrates can further comprise conventional components such ashousings, holders, adapters, and the like, and combinations thereof.

If desired, efficiency of binding and capture of biomaterials can beimproved by using a plurality of stacked or layered, functionalizedporous substrates (for example, functionalized porous membranes) as afilter element. Thus, a filter element can comprise one or more layersof functionalized porous substrate. The individual layers of the filterelement can be the same or different. The layers can vary in porosity,degree of grafting, and so forth. The filter element can furthercomprise an upstream prefilter layer and/or a downstream support layer.The individual layers can be planar or pleated, as desired.

Examples of suitable prefilter and support layer materials include anysuitable porous membranes of polypropylene, polyester, polyamide,resin-bonded or binder-free fibers (for example, glass fibers), andother synthetics (woven and nonwoven fleece structures); sinteredmaterials such as polyolefins, metals, and ceramics; yarns; specialfilter papers (for example, mixtures of fibers, cellulose, polyolefins,and binders); polymer membranes; and the like; and combinations thereof.

Useful articles for biomaterial capture or filtration applicationsinclude a filter cartridge comprising one or more of the above-describedfilter elements, a filter assembly comprising one or more of theabove-described filter elements and a filter housing, and the like. Thearticles can be used in carrying out a method of capture or removal of atarget biomaterial or biological species comprising (a) providing atleast one article comprising at least one above-described filterelement; and (b) allowing a moving biological solution containing atarget biomaterial to impinge upon the upstream surface of the filterelement for a time sufficient to effect binding of the targetbiomaterial.

EXAMPLES

Objects and advantages of this invention are further illustrated by thefollowing examples, but the particular materials and amounts thereofrecited in these examples, as well as other conditions and details,should not be construed to unduly limit this invention. These examplesare merely for illustrative purposes only and are not meant to belimiting on the scope of the appended claims.

Materials

Unless otherwise noted, all parts, percentages, ratios, etc., in theexamples and in the remainder of the specification are by weight. Unlessotherwise noted, all chemicals were obtained from, or are availablefrom, chemical suppliers such as Sigma-Aldrich Chemical Company, St.Louis, Mo.

VDM—vinyldimethylazlactone, SNPE, Inc, Princeton, N.J.MBA—methylenebisacrylamide, Sigma-Aldrich, Milwaukee, Wis.PEG200MA—polyethyleneglycol monomethacrylate, molecular weight (MW)about 200, Polysciences, Warrington, Pa.PEG400MA—polyethyleneglycol monomethacrylate, MW about 400,Polysciences, Warrington, Pa.IEM—2-isocyanatoethyl methacrylate, Showa Denko KK, Kanagawa, JapanS-BP—4-(3-sulfopropyloxy)benzophenone, sodium salt (which is awater-soluble benzophenone), prepared essentially as described inJapanese Patent No. 47040913 Teijin Ltd.)

Test Methods

Static Lysozyme Capacity Method for Functionalized Substrates

Functionalized substrates prepared as described in the Examples belowwere analyzed for static binding capacity by incubating one disk of thesubstrate in a solution of a test analyte overnight. The disk wasprepared by die-punching a 24-mm diameter disk from a sheet of thesubstrate. Each disk was placed in a 5 mL centrifuge tube with 4.5 mL ofchicken egg white lysozyme (Sigma-Aldrich, St. Louis, Mo.) challengesolution at a concentration of about 5.0 mg/mL in 10 mM MOPS(4-morpholinopropanesulfonic acid; Sigma-Aldrich, St. Louis, Mo.) bufferat pH 7.5. The tubes were capped and tumbled overnight (typically 14hours) on a rotating mixer (BARNSTEAD/THERMOLYN LABQUAKE™ Tube Shaker,obtained from VWR International, Eagan, Minn.). The resultingsupernatant solutions were analyzed using an ultraviolet-visible(UV-VIS) spectrometer (AGILENT™ 8453, Agilent Technologies, Santa Clara,Calif.) at 280 nanometers (nm) (with background correction applied at325 nm). The static binding capacity for each substrate was determinedby comparison to the absorbance of the starting lysozyme solution, andresults are reported in mg/mL (mg of protein bound/mL of membranevolume) as the average of three replicates.

Static BSA Capacity Method for Functionalized Substrates

Functionalized substrates prepared as described in the Examples belowwere analyzed for static binding capacity by incubating one disk of thesubstrate in a solution of a test analyte overnight. The disk wasprepared by die-punching a 24-mm diameter disk from a sheet of thesubstrate. Each disk was placed in a 5 mL centrifuge tube with 4.5 mL ofBSA (bovine serum albumin) challenge solution (Catalog # A-7906;Sigma-Aldrich, St. Louis, Mo.) at a concentration of about 3.0 mg/ml in25 millimolar TRIS (tris(hydroxymethyl)aminomethane; Sigma-Aldrich, St.Louis, Mo.) buffer, pH 8.0. The tubes were capped and tumbled overnight(typically 14 hours) on a rotating mixer (BARNSTEAD/THERMOLYN LABQUAKE™Tube Shaker, obtained from VWR International, Eagan, Minn.). Theresulting supernatant solutions were analyzed using a UV-VISspectrometer (AGILENT™ 8453, Agilent Technologies, Santa Clara, Calif.)at 279 nm (with background correction applied at 325 nm). The staticbinding capacity for each substrate was determined by comparison to theabsorbance of the starting BSA solution, and results are reported inmg/mL as the average of three replicates.

Static IgG Capacity Method for Functionalized Substrates (IgG Method 1)

Functionalized substrates prepared as described in the Examples belowwere analyzed for static binding capacity by incubating one disk of thesubstrate in a solution of a test analyte overnight. The disk wasprepared by die-punching a 24-mm diameter disk from a sheet of thesubstrate. Each disk was placed in a 5 mL centrifuge tube with 4.5 mL ofhuman IgG (Sigma-Aldrich, St. Louis, Mo.) challenge solution at aconcentration of about 5.5 mg/mL in 50 mM HEPES(N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid); Sigma-Aldrich,St. Louis, Mo.) buffer at pH 7.0. The tubes were capped and tumbledovernight (typically 14 hours) on a rotating mixer (BARNSTEAD/THERMOLYNLABQUAKE™ Tube Shaker, obtained from VWR International, Eagan, Minn.).The resulting supernate was poured off, and the disks were washed withHEPES buffer (4.5 mL) for 30 minutes on the rotating mixer. Theresulting supernate was poured off, and then the wash procedure wasrepeated. The resulting supernate was again poured off and was replacedby elution buffer (4.5 mL, 50 mM HEPES, 1 M NaCl, pH 7.0). The tubeswere tumbled on the rotating mixer for 30 minutes, then the resultingsupernate was analyzed using a UV-VIS spectrometer (AGILENT™ 8453,Agilent Technologies, Santa Clara, Calif.) at 280 nm (with backgroundcorrection applied at 325 nm). The static binding capacity for eachsubstrate was determined from the measured IgG concentration in thesupernate, and results are reported in mg/mL as the average of threereplicates.

Static IgG Capacity Method for Functionalized Substrates (IgG Method 2)

Functionalized substrates prepared as described in the Examples belowwere analyzed for static binding capacity by incubating one disk of thesubstrate in a solution of a test analyte overnight. The disk wasprepared by die-punching a 24-mm diameter disk from a sheet of thesubstrate. Each disk was placed in a 5 mL centrifuge tube with 4.5 mL ofhuman IgG (Equitech Bio, Kerrville, Tex.) challenge solution at aconcentration of about 2.0 mg/mL in 50 mM acetic acid/sodium acetatebuffer (Sigma-Aldrich, St. Louis, Mo.) with 40 mM NaCl at pH 4.5. Thetubes were capped and tumbled overnight (typically 14 hours) on arotating mixer (BARNSTEAD/THERMOLYN LABQUAKE™ Tube Shaker, obtained fromVWR International, Eagan, Minn.). The supernatant solutions wereanalyzed using a UV-VIS spectrometer (AGILENT™ 8453, AgilentTechnologies, Santa Clara, Calif.) at 280 nm (with background correctionapplied at 325 nm). The static binding capacity for each substrate wasdetermined by comparison to the absorbance of the starting IgG solution,and results are reported in mg/mL as the average of three replicates.

Graft Density and Ligand Efficiency

Nylon membrane substrates (nylon 66 membrane, single reinforced layernylon three-zone membrane, nominal pore size 1.8 μm, #080ZN, obtainedfrom 3M Purification, Inc., Meridan, Conn.) were equilibrated for aminimum of 18 hours in a low humidity chamber (Sanpia Dry Keeper,Sanplatec Corporation, available from VWR International) at a relativehumidity (RH) of 20-25 percent (%), prior to being grafted. Thesubstrates were removed from the low humidity chamber, weighedimmediately, and then subjected to a free radical grafting reaction asdescribed below for a variety of ligand functional group-containingmonomers. Following a washing and drying process (as described below),the substrates were again equilibrated in the low humidity chamber for aminimum of 18 hours, were removed from the chamber, and were reweighedimmediately to obtain a measurement of mass gain during the graftingreaction. The mass gain was subsequently utilized to estimate the numberof millimoles of monomer grafted to the substrate by dividing the massgain by the molecular weight of the monomer. Graft density was thennormalized by dividing by the original mass of the substrate andexpressed as millimoles of monomer grafted per gram of substrate(mmol/g). Ligand efficiency was expressed as the quotient of lysozymestatic capacity to graft density (capacity/mmol/g).

¹H NMR Analysis

Proton nuclear magnetic resonance (¹H NMR) analysis was carried outusing a nuclear magnetic resonance (NMR) spectrometer (BRUKER™ A500,obtained from Bruker Corp., Billerica, Mass.) in the solvents listed ineach example or table entry below. Splitting patterns in the ¹H-NMR dataare designated using the following abbreviations: s=singlet; br. s=broadsinglet; 2s=two singlets; d=doublet; dd=doublet of doublets; t=triplet;q=quartet; p=pentet; and m=multiplet.

Monomer Preparative Methods

Preparation of Ligand Functional Group-containing Monomers

Preparations of a variety of representative examples of ligandfunctional group-containing monomers are described below. Chemicalstructures are provided for several of the monomers. In some cases, anumbering scheme is also provided to illustrate the counting of spacergroup atoms.

Preparative Example 1 Preparation of the VDM Adduct of Glycine

Glycine (1.50 g, 0.02 mol) was charged to a 100 mL round bottom flask.Sodium hydroxide solution (1 N, 20 mL) was added to the flask. Theresulting mixture was stirred magnetically until dissolved and thencooled in an ice-water bath with continued stirring for 15 minutes. VDM(2.78 g, 2.5 mL, 0.02 mol) was added to the cooled mixture by syringe.The resulting mixture was stirred for 30 minutes with ice-bath coolingand then allowed to warm to room temperature over 30 minutes. ¹H-NMR(D₂O) δ 1.38; (s, 6H), 3.60; (s, 2H), 5.63; (d, 1H), 6.0-6.2; (m, 2H)indicated complete conversion to the desired acrylamide monomer as thesodium salt.

Preparative Examples 2-6

By procedures essentially analogous to that of Preparative Example 1(wherein m=1), additional monomers (having spacer groups of chainlength=5+m, wherein m varies, and having two hydrogen bond donors) wereprepared from the following aminoacids:

Prep. Ex. 2—β-alanine (m=2; number of spacer atoms=7): ¹H-NMR (D₂O) δ1.32; (s, 6H), 2.23; (t, 2H), 3.24; (t, 2H), 5.63; (m, 1H), 6.0-6.2; (m,2H)Prep. Ex. 3—γ-aminobutyric acid (m=3; number of spacer atoms =8): ¹H-NMR(D₂O) δ 1.34; (s, 6H), 1.59; (p, 2H), 2.04; (t, 2H), 3.05; (t, 2H),5.62; (d, 1H), 6.0-6.2; (m, 2H)Prep. Ex. 4—6-aminocaproic acid (m=5; number of spacer atoms=10): ¹H-NMR(D₂O) δ 1.14; (m, 2H), 1.34; (s and m, 8H), 1.41; (m, 2H), 2.02; (t,2H), 3.03; (t, 2H), 5.62; (d, 1H), 6.0-6.2; (m, 2H)Prep. Ex. 5—5-aminovaleric acid (m=4; number of spacer atoms=9): ¹H-NMR(D20) δ 1.33; (s and m, 10H), 2.04; (t, 2H), 3.05; (t, 2H), 5.63; (d,1H), 6.0-6.2; (m, 2H)

Prep. Ex. 6—11-aminoundecanoic acid (m=9; number of spacer atoms=14):¹H-NMR (CD₃OD) δ 1.26; (br. s, 12H), 1.45; (s and m, 8 H), 1.55; (m,2H), 2.12; (t, 2H), 3.13; (t, 2H),5.66; (d, 1H), 6.15-6.35; (m, 2H)

Preparative Examples 7-11

By procedures essentially analogous to that of Preparative Example 1,additional monomers (having spacer groups with the numbered chainlengths shown in the following structures and/or otherwise providedbelow) were prepared from the following aminoacids:

Prep. Ex. 7—diglycine (number of spacer atoms=9, and having threehydrogen bond donors): ¹H-NMR (D₂O) δ 1.37; (s, 6H), 3.66; (s, 2H),3.79; (s, 2 H), 5.66; (d, 1H), 6.12; (m, 2H)

Prep. Ex. 8—triglycine (number of spacer atoms=12, and having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.37; (s, 6H), 3.64; (s, 2H),3.82; (s, 2H), 3.88; (s, 2H) 5.66; (d, 1H), 6.11; (m, 2H)

Prep. Ex. 9—L-phenylalanine (number of spacer atoms=6, and having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.26; (s, 6H), 2.89; (m, 1H),2.95; (m, 1H), 4.30; (m, 1H), 5.62; (d, 1H), 6.00-6.10; (m, 2H),7.07-7.20; (m, 5H)Prep. Ex. 10—L-tryptophan (number of spacer atoms=6, and having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.12 and 1.14; (2s, 6H), 3.03; (m,1H), 3.14; (m, 1H), 4.34; (m, 1H), 5.52; (m, 1H), 5.93; (m, 2H),6.90-7.01; (m, 3H), 7.22; (d, 1H), 7.48; (d, 1H)Prep. Ex. 11—L-histidine (number of spacer atoms=6, and having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.23 and 1.25; (2s, 6H), 2.76;(dd, 1H), 2.91; (dd, 1H), 4.16; (m, 1H), 5.55; (m, 1H), 6.00; (m, 2H),6.68; (s, 1H), 7.43; (s, 1H)

Preparative Examples 12-15 Preparation of IEM Adducts of Aminoacids

Monomers (having spacer groups of chain length=7+m, wherein m varies)were prepared by essentially the procedure described in PreparativeExamples 1-4, except that VDM was replaced by IEM (3.1 g, 0.02 mol). Atthe end of each reaction, a small amount of colorless precipitate wasfiltered from the reaction mixture prior to use. ¹H-NMR verified theformation of the desired adducts as the sodium salts.

Prep. Ex. 12—glycine (m=1; number of spacer atoms=8; R =H; having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.79; (s, 3H), 3.33; (m, 2H),3.54; (s, 2H), 4.09; (m, 2 H), 5.59; (s, 1H), 5.99; (s, 1H)Prep. Ex. 13—β-alanine (m=2; number of spacer atoms=9; R=H; having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.78; (s, 3H), 2.22; (t, 2H),3.16; (t, 2H), 3.30; (t, 2H), 4.07; (t, 2H), 5.58; (s, 1H), 5.99; (s,1H)Prep. Ex. 14—γ-aminobutyric acid (m=3; number of spacer atoms=10; R =H;having two hydrogen bond donors): ¹H-NMR (D₂O) δ 1.57; (t, 2H), 1.78;(s, 3H), 2.05; (t, 2H), 2.95; (m, 2H), 3.31; (m, 2H), 4.08; (m, 2H),5.58; (s 1H), 5.99; (s, 1H)Prep. Ex. 15—6-aminocaproic acid (m=5; number of spacer atoms=12; R=H;having two hydrogen bond donors): ¹H-NMR (D₂O) δ 1.15; (m, 2H), 1.32;(m, 2H), 1.40; (m, 2H), 1.77; (s, 3H), 2.02; (m, 2H), 2.93; (m, 2H),3.30; (m, 2H), 4.07; (m, 2H), 5.58; (s, 1H), 5.99; (s, 1H)

Preparative Examples 16-21 Preparation of IEM Adducts of Aminoacids

Monomers (having spacer groups with the chain lengths provided below)were prepared by essentially the procedure described in PreparativeExamples 1-4, except that VDM was replaced by IEM (3.1 g, 0.02 mol). Atthe end of each reaction, a small amount of colorless precipitate wasfiltered from the reaction mixture prior to use. ¹H-NMR verified theformation of the desired adducts as the sodium salts.

Prep. Ex. 16—diglycine (number of spacer atoms=11; R=H; having threehydrogen bond donors): ¹H-NMR (D₂O) δ 1.79; (s, 3H) 3.34; (t, 2H), 3.65;(s, 2H), 3.72; (s, 2H), 4.11; (t, 2H), 5.59; (s, 1H), 6.00; (s, 1H)Prep. Ex. 17—triglycine (number of spacer atoms=14; R=H; having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.79; (s, 3H) 3.34; (t, 2H), 3.65;(s, 2H), 3.75; (s, 2H), 3.86; (s, 2H), 4.11; (t, 2H), 5.59; (s, 1H),6.00; (s, 1H)Prep. Ex. 18—sarcosine (number of spacer atoms=8; R=CH₃; having onehydrogen bond donor): ¹H-NMR (D₂O) δ 1.79; (s, 3H), 2.75; (s, 3H), 3.35;(t, 2H), 3.69; (s, 2H), 4.11; (t, 2H), 5.59; (d, 1H), 6.00; (d, 1H)Prep. Ex. 19—L-phenylalanine (number of spacer atoms=8; R=H; having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.74; (br. s, 3H), 2.73; (m, 1H),2.99; (m, 1H), 3.13; (m, 1H), 3.26; (m 1H), 3.90; (m, 2H), 4.17; (m,1H), 5.54; (m, 1H), 5.95; (m, 1H), 7.09; and 7.15; (m, 5H)Prep. Ex. 20—L-tryptophan (number of spacer atoms=8; R=H; having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.51; (s, 3H), 2.93; (m, 2H),3.10; (m, 2H), 3.67; (br. s, 2H), 4.27; (br. s, 1H), 5.24; (br. s, 1H),5.73; (br. s, 1H), 6.79; (m, 1H), 6.81; (m, 1H), 6.85; (s, 1H), 7.11;(m, 1H), 7.37; (m, 1H)Prep. Ex. 21—L-histidine (number of spacer atoms=8; R=H; having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.64; (s, 3H), 2.69; (m, 1H),2.86; (m, 1H), 3.14; (m, 1H), 3.22; (m, 1H), 3.91; (m, 2H), 4.08; (m,1H), 5.44; (m, 1H), 5.85; (s, 1H), 6.69; (s, 1H), 7.46; (s, 1H)

Preparative Example 22 Preparation of the Monomethacryloyloxyethyl Esterof Glutaric Acid

Glutaric anhydride (3.50 g, 0.03 mol) was charged to a 100 mL roundbottom flask, and dichloromethane (50 mL) was added to the flask. Theresulting mixture was stirred magnetically until the anhydridedissolved, and then 2-hydroxyethyl methacrylate (3.99 g, 0.03 mol) wasadded to the flask. The resulting mixture was stirred for 15 minutes andthen cooled in an ice-water bath to 0° C. Triethylamine (3.1 g, 4.3 mL,0.03 mol) was added by syringe to the stirring mixture, then4-dimethylaminopyridine (0.06 g, 0.03 mol) was also added. The resultingmixture was stirred for 2 hours with ice-bath cooling and was thenallowed to warm to room temperature over 30 minutes. The resultingmixture was stirred for an additional 12 hours at room temperature.

Excess solvent was then removed from the mixture by rotary evaporation.The resulting residue was dissolved in diethyl ether, and product wasextracted from the resulting solution into a saturated sodiumbicarbonate (3×50 mL) phase. The final pH of the phase (basic solution)was adjusted to 2 by adding 1N HCl. The product was extracted from theresulting acidic aqueous phase into diethyl ether (3×100 mL). Theresulting combined diethyl ether extract was then washed with brine anddried over Na2SO4. Solids were removed from the extract by filtration,and solvent was then removed by rotary evaporation to yield the productas a colorless liquid.

¹H-NMR (CDCl₃) δ 1.93; (t, 3H), 1.95; (m, 2H), 2.45; (m, 4H), 4.34; (m,4H), 5.59; (m, 1H), 6.12; (m, 1H) indicated complete conversion to thedesired monomer product (having 9 spacer atoms, as shown in thestructure above, but no hydrogen bond donors). A solution was preparedby dissolving the monomer (3.15 g, 0.013 mol) in 1 N sodium hydroxide(12.9 mL).

Preparative Example 23

2-Aminoethyltrimethylammonium chloride (Sigma-Aldrich, St. Louis, Mo.;5.1 g, 0.029 mol) was charged to a 100 mL round bottom flask anddissolved in sodium hydroxide solution (1N, 29 mL) with magneticstirring. The resulting solution was cooled in an ice-water bath for 10minutes, and then IEM (4.51 g, 0.029 mol) was added to the solution.Stirring was continued for 2 hours, and then a small amount of colorlessprecipitate was filtered. NMR analysis verified the formation of theexpected methacrylate adduct (having 9 spacer atoms, as shown in thestructure above, and two hydrogen bond donors). ¹H-NMR (D₂O) δ 1.80; (s,3H), 3.05; (s, 9H), 3.33; (m, 4H), 3.50; (m, 2H), 4.12; (t, 2H), 5.61;(s, 1H), 6.01; (s, 1H).

Preparative Example 24

The procedure of Preparative Example 23 was essentially repeated, exceptthat VDM (4.03 g, 0.029 mol) was used instead of IEM (to form monomerhaving 7 spacer atoms, as shown in the structure above, and two hydrogenbond donors). ¹H-NMR (D₂O) δ 1.30; (s, 6H), 3.01; (s, 9H), 3.30; (m,2H), 3.52; (m, 2H) 5.60; (m, 1H), 6.01; (m, 1H), 6.13; (m, 1H).

Preparative Examples 25-33

Monomers (having spacer groups with the numbered chain lengths shown inthe following structures and/or otherwise provided below) were preparedfrom various aminoacids by essentially the procedures described inPreparative Examples 1 and 12, using either VDM or IEM, respectively.Preparative Examples 25, 27, 29, 31, and 33 utilized 2N sodium hydroxideinstead of 1N sodium hydroxide. ¹H-NMR verified the formation of thedesired adducts as the sodium salts.

Prep. Ex. 25—L-aspartic acid/VDM (number of spacer atoms=6 and 7; havingtwo hydrogen bond donors): ¹H-NMR (D₂O) δ 1.32 & 1.33; (2s, 6H), 2.41;(m, 2H), 4.17; (m, 1H), 5.56; (d, 1H), 6.03; (m, 2H)Prep. Ex. 26—L-asparagine/VDM (number of spacer atoms=6; having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.31; (2s, 6H), 2.51; (m, 2H),4.25; (m, 1H), 5.56; (d, 1H), 6.03; (m, 2H)Prep. Ex. 27—L-glutamic acid/VDM (number of spacer atoms=6 and 8; havingtwo hydrogen bond donors): ¹H-NMR (D₂O) δ 1.37 & 1.38; (2s, 6H), 1.75;(m, 1H), 1.89; (m, 1H), 2.04; (m, 2H), 3.98; (m, 1H), 5.62; (d, 1H),6.05; (d, 1H), 6.17; (dd, 1H)Prep. Ex. 28—L-glutamine/VDM (number of spacer atoms=6; having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.31 & 1.33; (2s, 6H), 1.73; (m,1H), 1.92; (m, 1H), 2.08; (m, 2H), 3.98; (m, 1H), 5.58; (d, 1H), 6.05;(m, 2H)Prep. Ex. 29—L-aspartic acid/IEM (number of spacer atoms=8 and 9; havingtwo hydrogen bond donors): ¹H-NMR (D₂O) δ 1.73; (s, 3H), 2.26; (dd, 1H),2.44; (dd, 1H), 3.26; (m, 2H), 4.03; (t & m, 3H), 5.52; (s, 1H), 5.95;(s, 1H)

Prep. Ex. 30—L-asparagine/IEM (number of spacer atoms=8; having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.73; (s, 3H), 2.40; (dd, 1H),2.54; (dd, 1H), 3.26; (m, 2H), 4.03; (t, 2H), 4.15; (dd, 1H), 5.52; (s,1H), 5.94; (s, 1H)

Prep. Ex. 31—L-glutamic acid/IEM (number of spacer atoms=8 and 10;having two hydrogen bond donors): ¹H-NMR (D₂O) δ 1.63; (m 1H), 1.72; (s,3H), 1.80; (m, 1H), 2.00; (m, 2H), 3.26; (m, 2H), 3.79; (m, 1H), 4.03;(m, 2H), 5.51; (s, 1H), 5.93; (s, 1H)Prep. Ex. 32—L-glutamine/IEM (number of spacer atoms=8; having fourhydrogen bond donors): ¹H-NMR (D₂O) δ 1.72; (m, 1H), 1.73; (s, 3H),1.87; (m, 1H), 2.10; (m, 2H), 3.27; (m, 2H), 3.86; (m, 1H), 4.04; (t,2H), 5.52; (s, 1H), 5.94; (s, 1H)Prep. Ex. 33—iminodiacetic acid/IEM (number of spacer atoms=8; havingone hydrogen bond donor): ¹H-NMR (D₂O) δ 1.74; (s, 3H), 3.29; (t, 2H),3.64; (s, 4H), 4.03; (t, 2H), 5.53; (s, 1H), 5.96; (s, 1H)

Preparative Example 34

2-(Dimethylamino)ethanol (1.78 g, 0.02 mol, Alfa Aesar, Ward Hill, MA)was charged to a 25 mL glass vial. VDM (2.78 g, 0.02 mol) was added tothe vial, followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 2 drops;available from Sigma-Aldrich, St. Louis, Mo.). The vial was sealed, andthe contents of the vial were mixed by shaking. A mild exotherm ensued,and the exotherm was mediated by holding the vial under cold running tapwater for a few minutes. The vial was placed on a rocker for 1 hour, atwhich time NMR indicated complete conversion to the expectedacrylamidoester product (having 7 spacer atoms, and having one hydrogenbond donor). ¹H-NMR (CD₃OD) δ 1.47 (s, 6H), 2.26; (s, 6H), 2.60; (t,2H), 4.20; (t, 2H) 5.63; (dd, 1H), 6.21; (m, 2H).

Preparative Examples 35-37

By procedures essentially analogous to that of Preparative Example 34,additional monomers (having spacer groups of chain length=5+m, wherein mvaries, and having one hydrogen bond donor) were prepared from thefollowing aminoalcohols:

Prep. Ex. 35—3-(dimethylamino)-1-propanol (m=3; number of spaceratoms=8): ¹H-NMR (CD₃OD) δ 1.46; (s, 6H), 1.79; (m, 2H), 2.21; (s, 6H),2.35; (m, 2H), 4.11; (t, 2H), 5.64; (dd, 1H), 6.21; (m, 2H)Prep. Ex. 36—6-(dimethylamino)-1-hexanol (m=6; number of spaceratoms=11): (CD₃OD) δ 1.33; (m, 4H), 1.46 (s and m, 8H), 1.61; (m, 2H),2.22; (s, 6H), 2.29; (m, 2H0, 4.07; (t, 2H), 5.63; (dd, 1H), 6.20; (m,2H)Prep. Ex. 37—2-[2-(dimethylamino)ethoxy]ethanol (TCI, Ltd., Tokyo,Japan) (m=4 plus one ether oxygen in repeat unit; number of spaceratoms=10): ¹H-NMR (CD₃OD) δ 1.47; (s, 6H), 2.25; (s, 6H), 2.51; (t, 2H),3.57; (t, 2H), 3.62; (m, 2H), 4.21; (m, 2H), 5.63; (dd, 1H), 6.20; (m,2H)

Preparative Example 38

The monomer (a tertiary amine) from Preparative Example 34 (1.71 g, 7.5mmol) was dissolved in diethyl ether (25 mL) in a 100 mL round bottomedflask. Dimethylsulfate (0.945 g, 0.72 mL, 7.5 mmol) was added by syringeto the magnetically stirred solution. A colorless precipitate formedimmediately. The resulting mixture was allowed to stand overnight atroom temperature. The precipitate was broken up with a spatula,filtered, washed with ether, and dried under vacuum at room temperaturefor about 1 hour to provide an essentially quantitative yield of theexpected monomer (having 7 spacer atoms), a quaternary ammonium salt.¹H-NMR (D₂O) δ 1.38; (s, 6H), 3.05; (s, 9H), 3.62; (s and m, 5H), 4.47;(m, 2H), 5.67; (d, 1H), 6.10; (m, 2H). The monomer was dissolved indeionized water (10 mL) to prepare a monomer solution.

Preparative Examples 39-41

By procedures essentially analogous to that of Preparative Example 38,additional quaternary ammonium salt monomers were prepared from thefollowing tertiary amine monomers. In these examples, the precipitateswere not filtered, but rather the diethyl ether was removed by rotaryevaporation, the residue was dried under vacuum at room temperature for4 hours, and the dried residue was dissolved in deionized water (10 mL)to provide a monomer solution:

Prep. Ex. 39—Prep. Ex. 35 monomer (number of spacer atoms=8): ¹H-NMR(D₂O) δ 1.37; (s, 6H), 2.07; (m, 2H), 3.00; (s, 9H), 3.25; (m, 2H),3.61; (s, 3H), 4.13; (t, 2H), 5.67; (d, 1H), 6.12; (m, 2H)Prep. Ex. 40—Prep. Ex. 36 monomer (number of spacer atoms=11): ¹H-NMR(D₂O) δ 1.27; (m, 4H), 1.36; (s, 6H), 1.55; (m, 2H), 1.65; (m, 2H),2.97; (s, 9H), 3.17; (m, 2H), 3.62; (s, 3H), 4.03 (t, 2H), 5.65; (d,2H), 6.10; (m, 2H)Prep. Ex. 41—Prep. Ex. 37 monomer (number of spacer atoms=10): ¹H-NMR(D₂O) δ 1.37; (s, 6H), 3.06; (s, 9H), 3.45; (m, 2H), 3.62; (s, 3H),3.67; (m, 2H), 3.85; (m, 2H), 4.20; (m, 2H), 5.66; (d, 1H), 6.10; (m,2H)

Preparative Examples 42-43

Monomers were prepared from taurine (2-aminoethylsulfonic acid) byessentially the procedures described in Preparative Examples 1 and 12,using either VDM or IEM, respectively. ¹H-NMR verified the formation ofthe desired adducts as the sodium salts.

Prep. Ex. 42—taurine/VDM (number of spacer atoms=7, and having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.35; (s, 6H), 2.94; (t, 2H),3.45; (t, 2H) 5.64 (m, 1H), 6.10; (m, 2H)

Prep. Ex. 43—taurine/IEM (number of spacer atoms=9, and having twohydrogen bond donors): ¹H-NMR (D₂O) δ 1.75; (s, 3H), 2.88; (t, 2H),3.28; (t, 2H), 3.32; (t, 2H), 4.06; (t, 2H), 5.56; (2, 1H), 5.97; (s,1H)

Preparative Example 44

A monomer (having 8 spacer atoms and one hydrogen bond donor) wasprepared from IEM and N-benzylglycine (Sigma-Aldrich, St. Louis, Mo.) byessentially the procedure described in Preparative Example 12. ¹H-NMR(D₂O) δ 1.66; (s, 3H), 3.32; (t, 2H), 3.66; (s, 2H), 3.98; (t, 2H),4.32; (s, 2H), 5.48; (s, 1H), 5.86; (s, 1H), 7.04; (d, 2H), 7.13; (m,1H), 7.17; (m 2H).

Preparative Example 45

2-(Dimethylamino)ethanol (1.78 g, 0.02 mol) was charged to a 100 mLround bottom flask. Diethylether (20 mL) was added to the flask, and theresulting mixture was stirred magnetically until the alcohol dissolved.IEM (3.10 g, 0.02 mol) was added to the flask, and the resulting mixturewas stirred at room temperature for 3 hours to obtain the expectedurethane monomer product (having 9 spacer atoms and one hydrogen bonddonor). ¹H-NMR (CDCl₃) δ 1.86; (s, 3H), 2.19; (s, 6H), 2.46; (t, 2H),3.41; (m, 2H), 4.08; (t, 2H) 4.13; (t, 2H), 5.51; (s, 1H), 6.03; (s,1H).

Preparative Example 46(a)

2-[2-(Dimethylamino)ethoxy]ethanol (2.66 grams, 0.02 mol) was convertedto the corresponding methacrylate urethane monomer (having 12 spaceratoms and one hydrogen bond donor) by essentially the procedure ofPreparative Example 45. ¹H-NMR (CDCl₃) δ 1.82; (s, 3H), 2.14; (s, 6H),2.39; (t, 2H), 3.37; (m, 2H), 3.45; (t, 2H), 3.53; (m, 2H), 4.10; (m,4H), 5.38; (br. s, ca. 1H), 5.48; (s, 1H), 6.00 ;(s, 1H).

Preparative Example 46(b)

Preparative Example 46(a) was repeated. The reaction mixture was pouredinto a separatory funnel, followed by a 1N hydrochloric acid solution(20 mL). The resulting mixture was intimately mixed by shaking and thenallowed to phase separate, and the resulting lower aqueous phase wasseparated. NMR analysis indicated that the methacrylate urethane monomer(having 12 spacer atoms and one hydrogen bond donor) had been extractedinto the aqueous phase as the hydrochloride salt. ¹H-NMR (D₂O) δ 1.74;(s, 3H), 2.73; (s, 6H), 3.18; (t, 2H), 3.28; (t, 2H), 3.58; (m, 2H),3.67; (m, 2H), 4.06; (m, 4H), 5.55; (s, 1H), 5.95; (s, 1H).

Preparative Example 47

The reaction mixture from Preparative Example 46(a) was cooled in anice-water bath for 15 minutes. Dimethylsulfate (2.52 grams, 0.02 mole)was added to the mixture by syringe. An exothermic reaction ensued, andan oily precipitate appeared. The resulting mixture was stirred at roomtemperature for 1 hour, the resulting ether supernate was poured off,and the resulting residue was stripped on a rotary evaporator for anadditional 30 minutes to yield the expected quaternary ammonium saltmonomer (having 12 spacer atoms and one hydrogen bond donor) as aviscous oil. ¹H-NMR (D₂O) δ 1.74; (s, 3H), 3.00; (s, 9H), 3.28; (t, 2H),3.41; (m, 2H), 3.56; (s, 3H), 3.58; (m, 2H), 3.81; (m, 2H), 4.06; (m,4H), 5.56; (s, 1H), 5.95; (s, 1H).

Preparative Example 48

A 250 mL round bottom flask was charged with ethanol (50 mL) andsuccinic anhydride (2.00 grams, 0.02 mol). The resulting mixture wasstirred magnetically until the anhydride dissolved. 4-Aminobenzylamine(2.44 grams, 0.02 mol) was added to the flask. The resulting mixture wasstirred for 5 days. An off-white solid precipitated and was filtered,washed with diethylether, and dried under vacuum. ¹H-NMR (D₂O) δ 2.32;(m, 4H), 4.13; (s, 2H), 6.93; (d, 2H) 7.09; (d, 2H).

A portion of the dried product (0.5 grams, 2.25 mmol) was dissolved in2.25 grams of 1N NaOH, and the resulting solution was stirredmagnetically and cooled in an ice-water bath for 15 minutes. IEM (0.35grams, 2.25 mmol) was added to the solution. The resulting mixture wasstirred and allowed to warm to room temperature over 4.5 hours. Aresulting small amount of colorless precipitate was filtered. NMRanalysis indicated clean formation of the desired carboxylic acidmethacrylate monomer (having 16 spacer atoms, as shown in the abovestructure, and three hydrogen bond donors). ¹H-NMR (D₂O) δ 1.71; (s,3H), 2.30; (m, 4H), 3.31; (t, 2H), 4.05; (t, 2H), 4.13; (s, 2H), 5.50;(s, 1H), 5.93; (s, 1H), 7.04; (m, 4H).

Preparative Comparative Example 1

Sodium acrylate (Sigma-Aldrich, St. Louis, Mo.; 4.70 g, 0.05 mol) wasdissolved in deionized water (50 mL) to prepare a monomer (having zerospacer atoms) solution essentially equivalent in concentration to thoseof Preparative Examples 1-8.

Preparative Comparative Example 2

N-acryloyl-2-methylalanine, prepared essentially according to Example 7of U.S. Pat. No. 4,157,418 (Heilmann) (7.85 g, 0.05 mol) was dissolvedin sodium hydroxide solution (1 N, 50 mL) to prepare a monomer (having 3spacer atoms and one hydrogen bond donor) solution essentiallyequivalent in concentration to those of Preparative Examples 1-8.

Preparative Comparative Example 3

N-acryloylglycine, prepared essentially according to Example 1 of U.S.Pat. No. 4,157,418 (Heilmann) (6.45 g., 0.05 mol) was dissolved insodium hydroxide solution (1 N, 50 mL) to prepare a monomer (having 3spacer atoms and one hydrogen bond donor) solution essentiallyequivalent in concentration to those of Preparative Examples 1-8.

Preparative Comparative Example 4

2-Carboxyethylacrylate (Sigma-Aldrich, St. Louis, Mo.; 7.2 g, 0.05 mol)was dissolved in sodium hydroxide solution (1 N, 50 mL) to prepare amonomer (having 4 spacer atoms) solution essentially equivalent inconcentration to those of Preparative Examples 1-8.

Preparative Comparative Example 5

Methacrylic acid (4.30 g, 0.05 mol) was dissolved in sodium hydroxidesolution (1N, 50 mL) to prepare a monomer (having zero spacer atoms)solution essentially equivalent in concentration to those of PreparativeExamples 12-22.

Preparative Comparative Example 6

[2-(Methacryloyloxy)ethyl]trimethylammonium chloride (Sigma-Aldrich, St.Louis, Mo.; 13.85 g of a 75 percent (%) by weight solution in water) wasdiluted with deionized water (36 mL) to prepare a monomer (having 4spacer atoms) solution of essentially equivalent molar concentration tothat of Preparative Example 23.

Preparative Comparative Example 7

The solution from Preparative Comparative Example 6 (3.75 grams) wasdiluted with deionized water (1.25 grams).

Preparative Comparative Example 8

[3-(Methacryloylamino)propyl]trimethylammonium chloride (Sigma-Aldrich,St. Louis, Mo.; 22.07 g of a 50 percent (%) by weight solution in water)was diluted with deionized water (27.9 g) to prepare a monomer (having 5spacer atoms and one hydrogen bond donor) solution of essentiallyequivalent molar concentration to that of Preparative Example 24.

Preparative Comparative Example 9

The solution from Preparative Comparative Example 8 (3.75 grams) wasdiluted with deionized water (1.25 grams).

Preparative Comparative Example 10

3-(Acrylamidopropyl)trimethylammonium chloride (Sigma-Aldrich, St.Louis, Mo.; 2.07 g of a 75 percent (%) by weight solution in water) wasdiluted with deionized water (9.5 g) to prepare a monomer (having 5spacer atoms and one hydrogen bond donor) solution of essentiallyequivalent molar concentration to those of Examples 38-41.

Preparative Comparative Example 11

2-Acrylamido-2-methyl-l-propanesulfonic acid, sodium salt(Sigma-Aldrich, St. Louis, Mo.; 41.34 g of a 50 percent (%) by weightsolution in water) was diluted with deionized water (79.3 g) to preparea monomer (having 4 spacer atoms and one hydrogen bond donor) solutionof essentially equivalent concentration to those of Preparative Examples42 and 43.

Preparative Comparative Example 12

N-acryloylphenylalanine, prepared essentially according to Example 10 ofU.S. Pat. No. 4,157,418 (Heilmann) (10.95 g, 0.05 mol) was dissolved insodium hydroxide solution (1 N, 50 mL) to prepare a monomer (having 3spacer atoms and one hydrogen bond donor) solution essentiallyequivalent in concentration to those of Preparative Examples 1-8.

Examples 1-6 and Comparative Examples C-1-C-4

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Examples 1-4, 7, and 8 and Preparative Comparative Examples1-4 (3.75 g) with deionized water (1.25 g) and S-BP (250 μL of a 0.1g/mL solution in deionized water). For each coating solution, a nylonmembrane substrate (9 cm×12 cm; nylon 66 membrane, single reinforcedlayer nylon three-zone membrane, nominal pore size 1.8 μm, #080ZN,obtained from 3M Purification, Inc., Meridan, Conn.) was placed on asheet of polyester film, and approximately 4.5 mL of coating solutionwas pipetted onto the top surface of the substrate. The coating solutionwas allowed to soak into the substrate for about 1 minute, and then asecond sheet of polyester film was placed on top of the substrate. A2.28 kg cylindrical weight was rolled over the top of the resultingthree-layer sandwich to squeeze out excess coating solution. Ultraviolet(UV)-initiated grafting was conducted by irradiating the sandwich usinga UV stand (Classic Manufacturing, Inc., Oakdale, Minn.) equipped with18 bulbs (Sylvania RG2 40W F40/350BL/ECO, 10 above and 8 below thesubstrate, 1.17 meters (46 inches) long, spaced 5.1 cm (2 inches) oncenter), with an irradiation time of 15 minutes. The polyester sheetswere removed, and the resulting functionalized substrate was placed in a250 mL polyethylene bottle. The bottle was filled with 0.9 percent (%)saline, sealed, and shaken for 30 minutes to wash off any residualmonomer or ungrafted polymer. The saline was poured off, and thefunctionalized substrate was washed for another 30 minutes with freshsaline solution and then washed for 30 minutes with deionized water andallowed to dry.

The functionalized substrate was tested for graft density and staticlysozyme binding capacity, from which ligand efficiency was calculated.Results are reported in Table 1(a) below for each coating solution. Inorder to minimize the effects of experimental variability, the substratefunctionalization procedure was often repeated and the results of thenumber of repetitions (N) averaged to provide the reported values.

TABLE 1(a) Lysozyme Ligand Number Graft Static Efficiency of ExampleDensity Capacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g)N Atoms 1 Prep. Ex. 1 0.64 151 237 3 6 2 Prep. Ex. 2 0.62 176 281 3 7 3Prep. Ex. 3 0.49 139 283 3 8 4 Prep. Ex. 4 0.61 177 291 3 10 5 Prep. Ex.7 0.49 141 288 2 9 6 Prep. Ex. 8 0.44 145 329 2 12 C-1 Prep. Comp. 0.4440 90 3 0 Ex. 1 C-2 Prep. Comp. 0.75 118 159 3 3 Ex. 2 C-3 Prep. Comp.0.81 152 190 3 3 Ex. 3 C-4 Prep. Comp. 0.57 102 179 2 4 Ex. 4Several of the functionalized substrates were also tested for static IgGcapacity (IgG Method 1) and corresponding ligand efficienciescalculated. Results are shown in Table 1(b).

TABLE 1(b) IgG Static Capacity Ligand Efficiency Example No. (mg/mL)(capacity/mmol/g) 1 134 210 2 157 247 3 169 357 4 169 284 C-1 123 148C-2 126 172 C-3 63 165

Examples 7-14 and Comparative Example C-5

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Examples 12-18 and 22 and Preparative Comparative Example 5(3.75 g) with deionized water (1.25 g) and S-BP (250 μL of a 0.1 g/mLsolution in deionized water). Nylon membrane substrates were coated,grafted, washed, and evaluated essentially as described in Example 1.Results are shown in Table 2 below.

TABLE 2 Lysozyme Ligand Number Graft Static Efficiency of ExampleDensity Capacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g)N Atoms 7 Prep. Ex. 12 0.51 119 232 3 8 8 Prep. Ex. 13 0.50 122 248 2 99 Prep. Ex. 14 0.53 133 250 3 10 10 Prep. Ex. 15 0.56 126 223 3 12 11Prep. Ex. 16 0.55 145 264 2 11 12 Prep. Ex. 17 0.52 157 302 2 14 13Prep. Ex. 18 0.56 120 216 2 8 14 Prep. Ex. 22 0.59 112 191 2 9 C-5 Prep.Comp. 0.36 36 102 3 0 Ex. 5

Example 15 Preparation and Grafting of the Poly(ethyleneglycol)(200)monomethylacrylate Ester of Glutaric Acid

Glutaric anhydride (3.50 g, 0.03 mol) was charged to a 100 mL roundbottom flask, and dichloromethane (50 mL) was added to the flask. Theresulting mixture was stirred magnetically until the anhydridedissolved, and then PEG200MA (6.70 g, 0.025 mol) was added to the flask.The resulting mixture was stirred for 15 minutes and then cooled in anice-water bath to 0° C. Triethylamine (3.1 g, 4.3 mL, 0.03 mol) wasadded by syringe to the stirring mixture, then 4-dimethylaminopyridine(0.06 g, 0.03 mol) was also added. The resulting mixture was stirred for2 hours with ice-bath cooling and then allowed to warm to roomtemperature over 30 minutes. The resulting mixture was stirred for anadditional 12 hours at room temperature.

Excess solvent was removed from the mixture by rotary evaporation. Theresulting residue was dissolved in diethyl ether, and product wasextracted from the resulting solution into a saturated sodiumbicarbonate (3×50 mL) phase. The final pH of the phase (basic solution)was adjusted to 2 by adding 1N HCl. Then, the product was extracted fromthe resulting acidic aqueous phase into a diethyl ether (3×100 mL)phase, which was then washed with brine and dried over Na2SO4. Excesssolvent was removed from the diethyl ether phase by rotary evaporationto yield the product as a colorless liquid. ¹H-NMR (DMSO-d6) δ 1.71,1.88, 2.24, 2.33, 3.49, 3.63, 4.12, 4.20, 4.28, 5.68, 6.02 indicatedcomplete conversion to the desired carboxylic acid monomer havingapproximately 18 spacer atoms and no hydrogen bond donors.

When the monomer was grafted to a nylon membrane substrate essentiallyas described in Example 1, the resulting functionalized substratedisplayed lysozyme binding capacity similar to that of Example 14.

Examples 16-19 and Comparative Examples C-6-C-9

Coating solutions were prepared as follows. For Examples 16 and 18 andComparative Examples C-6 and C-8, the monomer solutions from PreparativeExamples 23 and 24 and Comparative Examples 6 and 8 (5 grams) were eachmixed with S-BP (250 μL of a 0.1 g/mL solution in deionized water). ForExamples 17 and 19, each monomer solution (3.75 grams) was diluted withdeionized water (1.25 g) prior to mixing with the S-BP solution. ForComparative Examples C-7 and C-9, the S-BP solution was added directlyto the respective monomer solutions before coating. Nylon membranesubstrates were coated, grafted, washed, and evaluated essentially asdescribed in Example 1, except that BSA static binding capacity wasmeasured. Results are shown in Table 3.

TABLE 3 BSA Ligand Number Graft Static Efficiency of Example DensityCapacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g) N Atoms16 Prep. Ex. 23 0.62 133 215 1 9 17 Prep. Ex. 23 0.44 96 219 1 9 18Prep. Ex. 24 0.60 154 258 1 7 19 Prep. Ex. 24 0.42 132 316 1 7 C-6 Prep.Comp. 0.89 146 164 1 4 Ex. 6 C-7 Prep. Comp. 0.57 94 166 1 4 Ex. 7 C-8Prep. Comp. 1.03 137 133 1 5 Ex. 8 C-9 Prep. Comp. 0.54 78 143 1 5 Ex. 9

Examples 20-23 and Comparative Example C-10

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Examples 1-4 and Preparative Comparative Example 2 (20 g)with MBA (1.2 mL of a 0.1 g/mL solution in methanol), PEG400MA (1.6 g),and S-BP (1.0 mL of a 0.1 g/mL solution in deionized water). Nylonmembrane substrates were coated, grafted, and washed essentially asdescribed in Example 1, except that the time of UV irradiation was only10 minutes. The resulting functionalized substrates were tested forstatic lysozyme and IgG (IgG Method 2) binding capacities, and theresults are shown in Table 4.

TABLE 4 Lysozyme Static IgG Static Capacity Capacity Example No. Monomer(mg/mL) (mg/mL) 20 Prep. Ex. 1 215 213 21 Prep. Ex. 2 276 242 22 Prep.Ex. 3 309 243 23 Prep. Ex. 4 354 119 C-10 Prep. Comp. Ex. 2 198 130

Examples 24-32

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Examples 25-33 (3.75 g) with deionized water (1.25 g) andS-BP (250 μL of a 0.1 g/mL solution in deionized water). Nylon membranesubstrates were coated, grafted, washed, and evaluated essentially asdescribed in Example 1. Results are shown in Table 5(a).

TABLE 5(a) Lysozyme Ligand Number Graft Static Efficiency of ExampleDensity Capacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g)N Atoms 24 Prep. Ex. 25 0.48 73 151 1 6, 7 25 Prep. Ex. 26 0.57 89 156 16 26 Prep. Ex. 27 0.58 124 213 1 6, 8 27 Prep Ex. 28 0.50 95 189 1 6 28Prep. Ex. 29 0.41 58 139 1 8, 9 29 Prep. Ex. 30 0.42 129 306 1 8 30Prep. Ex. 31 0.38 75 199 1 8, 10 31 Prep. Ex. 32 0.48 139 292 1 8 32Prep. Ex. 33 0.43 87 203 1 8

The resulting functionalized substrates were also tested for static IgGcapacities (IgG Method 1) and corresponding ligand efficienciescalculated. Results, showing relatively high capacities and efficienciesfor IgG capture, are shown in Table 5(b).

TABLE 5(b) IgG Static Capacity Ligand Efficiency Example No. (mg/mL)(capacity/mmol/g) 24 183 381 25 173 303 26 169 290 27 188 373 28 187 45329 179 426 30 204 541 31 196 412 32 192 449

Examples 33-36 and Comparative Example C-11

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Examples 38-41 and Preparative Comparative Example 10 (5.0g) with S-BP (250 μL of a 0.1 g/mL solution in deionized water). Nylonmembrane substrates were coated, grafted, washed, and evaluatedessentially as described in Example 16. Results are shown in Table 6.

TABLE 6 BSA Static Capacity Number of Example No. Monomer (mg/mL) SpacerAtoms 33 Prep. Ex. 38 145 7 34 Prep. Ex. 39 166 8 35 Prep. Ex. 40 161 1136 Prep. Ex. 41 168 10 C-11 Prep. Comp. Ex. 10 121 5

Examples 37-38 and Comparative Example C-12

Coating solutions were prepared as follows. For Examples 37 and 38 andComparative Example 12, the monomer solutions from Preparative Examples42 and 43 and Preparative Comparative Example 11 (5 grams) were eachmixed with S-BP (250 μL of a 0.1 g/mL solution in deionized water).Nylon membrane substrates were coated, grafted, washed, and evaluatedessentially as described in Example 1. Results are shown in Table 7.

TABLE 7 Lysozyme Ligand Number Graft Static Efficiency of ExampleDensity Capacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g)N Atoms 37 Prep. Ex. 42 0.41 147 356 1 7 38 Prep. Ex. 43 0.50 153 303 19 C-12 Prep. Comp. 1.19 143 120 1 4 Ex. 11

Example 39

The monomer solution of Preparative Example 48 was formulated, coated,grafted, washed, and evaluated essentially as described in Example 1. Asthe average of three trials, the resulting substrates functionalizedwith this monomer, having a spacer length of 16 atoms, exhibited a graftdensity of 1.01 mmol/g, lysozyme static capacity of 195 mg/mL, and aligand efficiency of 193.

Example 40

The monomer solution of Preparative Example 46(b) (3.75 g) was mixedwith deionized water (1.25 g) and S-BP (250 μL of a 0.1 g/mL solution indeionized water). Nylon membrane substrates were coated, grafted,washed, and dried essentially as described in Example 1. Static BSAcapacity was measured as described above, except that the TRIS bufferwas replaced by 10 millimolar MOPS (3-(N-morpholino)propanesulfonicacid; Sigma-Aldrich, St. Louis, Mo.), pH 7.5. Under these conditions,graft density was 0.75 mmol/g, BSA capacity was 74.9 mg/mL, and ligandefficiency was 159.

Example 41 and Comparative Example C-13

Coating solutions were prepared by mixing monomer solutions from each ofPreparative Example 9 and Preparative Comparative Example 12 (3.75 g)with deionized water (1.25 g) and S-BP (250 μL of a 0.1 g/mL solution indeionized water). Nylon membrane substrates were coated, grafted,washed, and evaluated essentially as described in Example 1. Results areshown in Table 8.

TABLE 8 Lysozyme Ligand Number Graft Static Efficiency of ExampleDensity Capacity (capacity/ Spacer No. Monomer (mmol/g) (mg/mL) mmol/g)N Atoms 41 Prep. Ex. 9 0.56 113 204 2 6 C-13 Prep. Comp. 0.41 58 143 3 3Ex. 12

Example 42 Preparation and Grafting of Phosphonate Monomer

Ex.42(a)—2-Dimethoxyphosphorylethyl-2-methyl-2-(prop-2-enoylamino)propanoate

A solution of VDM (1.63 g, 11.7 mmol) in 10 mL of anhydrous methylenechloride was treated with hydroxyethyl dimethylphosphonate (90 percent(%) purity, 2.00 g, 11.7 mmol) and one drop of1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; available from Sigma-Aldrich,St. Louis, Mo.). After stirring overnight, the resulting mixture wasdiluted with methylene chloride and washed successively with saturated5% NaH₂PO₄, H₂O, and brine. The resulting organic portion was dried overNa₂SO₄ and filtered. A small amount (approximately 3 mg) of2,6-di-tert-butyl-4-methylphenol (BHT; Sigma-Aldrich, St. Louis, Mo.)was added to the filtered portion, and the resulting solution wasconcentrated under reduced pressure at ambient temperature (about 23°C.) to give 3.06 g of colorless liquid. ¹H NMR (CDCl₃, 500 MHz)□6.35;(br s, 1H), 6.27; (dd, J=1.4, 17.0 Hz, 1H), 6.12; (dd, J=10.2, 17.0 Hz,1H), 5.64; (dd, J=1.4, 10.2 Hz, 1H), 4.39; (dt, J=13.3, 7.3 Hz, 2H),3.77; (d, J=10.9 Hz, 6H), 2.19; (dt, J=18.8, 7.3 Hz, 2H), 1.60; (s, 6H).

Ex. 42(b)—2[2-Methyl-2-(prop-2-enoylamino)propanoyl]oxyethylphosphonicAcid, Sodium Salt

The above-prepared colorless liquid (3.06 g, 10.4 mmol) was dissolved in10 mL of anhydrous methylene chloride and treated with trimethylsilylbromide (3.24 g, 21.2 mmol). The resulting solution was stirred for 3hours and then concentrated under reduced pressure at ambienttemperature (about 23° C.). The resulting oil was then concentrated frommethanol twice to give 2.77 g of colorless syrup. ¹H NMR (D₂O, 500MHz)□6.15; (dd, J=10.1, 17.1 Hz, 1H), 6.06; (dd, J=1.4, 17.1 Hz, 1H),5.55; (dd, J=1.4, 10.1 Hz, 1H), 4.25; (dt, J =15.1, 7.1 Hz, 2H), 2.09;(dt, J=18.2, 7.1 Hz, 2H), 1.38 (s, 6H). The syrup was dissolved in 1Nsodium hydroxide (10.45 mL) to prepare a monomer solution comprising themonomer shown above, having seven spacer atoms and one hydrogen bonddonor. The monomer solution was formulated, coated, grafted, washed, andevaluated essentially as described in Example 1. The resulting substratefunctionalized with this monomer exhibited a graft density of 0.46mmol/g, a lysozyme static capacity of 179 mg/mL, and a ligand efficiencyof 391.

The referenced descriptions contained in the patents, patent documents,and publications cited herein are incorporated by reference in theirentirety as if each were individually incorporated. Variousunforeseeable modifications and alterations to this invention willbecome apparent to those skilled in the art without departing from thescope and spirit of this invention. It should be understood that thisinvention is not intended to be unduly limited by the illustrativeembodiments and examples set forth herein and that such examples andembodiments are presented by way of example only, with the scope of theinvention intended to be limited only by the claims set forth herein asfollows.

1-23. (canceled)
 24. A free radically polymerizable compound consistingof (a) at least one monovalent ethylenically unsaturated group, (b) atleast one monovalent ligand functional group selected fromphosphorus-containing acidic groups, boron-containing acidic groups, andsalts thereof, and (c) a multivalent spacer group that is directlybonded to the monovalent groups so as to link at least one ethylenicallyunsaturated group and at least one ligand functional group by a chain ofat least six catenated atoms, said monomer of the formula:CH₂═CR¹—C(═O)—X—R²—[Z—R²]_(n)—L wherein R¹ is selected from hydrogen,alkyl, cycloalkyl, aryl, and combinations thereof; each R² isindependently selected from hydrocarbylene, heterohydrocarbylene, andcombinations thereof; X is —O— or —NR³—, where R³ is selected fromhydrogen, hydrocarbyl, heterohydrocarbyl, and combinations thereof; Z isheterohydrocarbylene comprising at least one hydrogen bond donor, atleast one hydrogen bond acceptor, or a combination thereof; n is 1; andL is a heteroatom-containing group comprising at least one monovalentligand functional group selected from acidic groups, basic groups otherthan guanidino, and salts thereof.
 25. The compound of claim 24, whereinsaid ligand functional group is selected from phosphono, phosphato,boronato, and combinations thereof.